Modulators of atp-binding cassette transporters

ABSTRACT

Compounds of the present invention and pharmaceutically acceptable compositions thereof, are useful as modulators of ATP-Binding Cassette (“ABC”) transporters or fragments thereof, including Cystic Fibrosis Transmembrane Conductance Regulator (“CFTR”). The present invention also relates to methods of treating ABC transporter mediated diseases using compounds of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/557,043, filed Nov. 8, 2011, and U.S. Provisional ApplicationSer. No. 61/610,257, filed Mar. 13, 2012, the entire contents of whichare incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to modulators of ATP-Binding Cassette(“ABC”) transporters or fragments thereof, including Cystic FibrosisTransmembrane Conductance Regulator (“CFTR”), compositions thereof andmethods therewith. The present invention also relates to methods oftreating ABC transporter mediated diseases using such modulators.

BACKGROUND OF THE INVENTION

ABC transporters are a family of membrane transporter proteins thatregulate the transport of a wide variety of pharmacological agents,potentially toxic drugs, and xenobiotics, as well as anions. ABCtransporters are homologous membrane proteins that bind and use cellularadenosine triphosphate (ATP) for their specific activities. Some ofthese transporters were discovered as multidrug resistance proteins(like the MDR1-P glycoprotein, or the multidrug resistance protein,MRP1), defending malignant cancer cells against chemotherapeutic agents.To date, 48 ABC Transporters have been identified and grouped into 7families based on their sequence identity and function.

ABC transporters regulate a variety of important physiological roleswithin the body and provide defense against harmful environmentalcompounds. Because of this, they represent important potential drugtargets for the treatment of diseases associated with defects in thetransporter, prevention of drug transport out of the target cell, andintervention in other diseases in which modulation of ABC transporteractivity may be beneficial.

One member of the ABC transporter family commonly associated withdisease is the cAMP/ATP-mediated anion channel, CFTR. CFTR is expressedin a variety of cells types, including absorptive and secretoryepithelia cells, where it regulates anion flux across the membrane, aswell as the activity of other ion channels and proteins. In epitheliacells, normal functioning of CFTR is critical for the maintenance ofelectrolyte transport throughout the body, including respiratory anddigestive tissue. CFTR is composed of approximately 1480 amino acidsthat encode a protein made up of a tandem repeat of transmembranedomains, each containing six transmembrane helices and a nucleotidebinding domain. The two transmembrane domains are linked by a large,polar, regulatory (R)-domain with multiple phosphorylation sites thatregulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in CysticFibrosis (“CF”), the most common fatal genetic disease in humans. CysticFibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia leads to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, >1000 disease causingmutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺-K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl− channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

In addition to Cystic Fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. These include, but are not limited to, emphysema, chronicobstructive pulmonary disease (COPD), dry eye disease, and Sjögren'sSyndrome.

COPD is characterized by airflow limitation that is progressive and notfully reversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such asCystic Fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Modulators of CFTR activity mayhydrate the various organs afflicted by the disease and help to elevatethe associated symptoms.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases. The two ways that the ER machinery can malfunction is eitherby loss of coupling to ER export of the proteins leading to degradation,or by the ER accumulation of these defective/misfolded proteins [AridorM, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et al.,Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21,pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198(1999)]. The diseases associated with the first class of ER malfunctionare Cystic fibrosis (due to misfolded ΔF508-CFTR as discussed above),emphysema (due to al-antitrypsin; non Piz variants), Hereditaryhemochromatosis, Coagulation-Fibrinolysis deficiencies, such as ProteinC deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses (due to Lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-Hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus (due to Insulinreceptor), Laron dwarfism (due to Growth hormone receptor),Myleoperoxidase deficiency, Primary hypoparathyroidism (due toPreproparathyroid hormone), Melanoma (due to Tyrosinase). The diseasesassociated with the latter class of ER malfunction are Glycanosis CDGtype 1, emphysema (due to al-Antitrypsin (PiZ variant), Congenitalhyperthyroidism, Osteogenesis imperfecta (due to Type I, II, IVprocollagen), Hereditary hypofibrinogenemia (due to Fibrinogen), ACTdeficiency (due to a 1-Antichymotrypsin), Diabetes insipidus (DI),Neurophyseal DI (due to Vasopvessin hormone/V2-receptor), Neprogenic DI(due to Aquaporin II), Charcot-Marie Tooth syndrome (due to Peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, Amyotrophic lateral sclerosis, Progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders asuch as Huntington, Spinocerebullar ataxia type I, Spinal andbulbar muscular atrophy, Dentatorubal pallidoluysian, and Myotonicdystrophy, as well as Spongiform encephalopathies, such as HereditaryCreutzfeldt-Jakob disease (due to Prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A) andStraussler-Scheinker syndrome (due to Prp processing defect).

In addition to up-regulation of CFTR activity, reducing anion secretionby CFTR modulators may be beneficial for the treatment of secretorydiarrheas, in which epithelial water transport is dramatically increasedas a result of secretagogue activated chloride transport. The mechanisminvolves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the major consequencesof diarrheal diseases, resulting from excessive chloride transport arecommon to all, and include dehydration, acidosis, impaired growth anddeath.

Acute and chronic diarrheas represent a major medical problem in manyareas of the world. Diarrhea is both a significant factor inmalnutrition and the leading cause of death (5,000,000 deaths/year) inchildren less than five years old.

Secretory diarrheas are also a dangerous condition in patients ofacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). 16 million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Diarrhea in barn animals and pets such as cows, pigs and horses, sheep,goats, cats and dogs, also known as scours, is a major cause of death inthese animals. Diarrhea can result from any major transition, such asweaning or physical movement, as well as in response to a variety ofbacterial or viral infections and generally occurs within the first fewhours of the animal's life.

The most common diarrhea causing bacteria is enterotoxogenic E-coli(ETEC) having the K99 pilus antigen. Common viral causes of diarrheainclude rotavirus and coronavirus. Other infectious agents includecryptosporidium, giardia lamblia, and salmonella, among others.

Symptoms of rotaviral infection include excretion of watery feces,dehydration and weakness. Coronavirus causes a more severe illness inthe newborn animals, and has a higher mortality rate than rotaviralinfection. Often, however, a young animal may be infected with more thanone virus or with a combination of viral and bacterial microorganisms atone time. This dramatically increases the severity of the disease.

Accordingly, there is a need for modulators of an ABC transporteractivity, and compositions thereof, that can be used to modulate theactivity of the ABC transporter in the cell membrane of a mammal.

There is a need for methods of treating ABC transporter mediateddiseases using such modulators of ABC transporter activity.

There is a need for methods of modulating an ABC transporter activity inan ex vivo cell membrane of a mammal.

There is a need for modulators of CFTR activity that can be used tomodulate the activity of CFTR in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases usingsuch modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are useful asmodulators of ABC transporter activity, particularly CTFR activity.These compounds have the general formula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein        independently for each occurrence:    -   Y is OH or NH; and    -   X is CO₂J;        -   wherein J is H or C₁-C₆ alkyl;    -   R is H, OH, OCH₃ or two R taken together form —OCH₂O— or        —OCF₂O—;    -   R₁ is H or up to two C₁-C₆ alkyl;    -   R₂ is H or halo; and    -   R₃ is H or C₁-C₆ alkyl;    -   or Y and X combine to form a compound of formula II:

-   -   or a pharmaceutically acceptable salt thereof, wherein        independently for each occurrence:    -   R is H, OH, OCH₃ or two R taken together form —OCH₂O— or        —OCF₂O—;    -   R₁ is H or up to two C₁-C₆ alkyl;    -   R₂ is H or halo;    -   R₃ is H or C₁-C₆ alkyl;    -   Y is O or NR₄; and    -   R₄ is H or C₁-C₆ alkyl.

The invention also provides methods for preparing compounds of formula Iand II.

These compounds and pharmaceutically acceptable compositions thereof areuseful for treating or lessening the severity of a variety of diseases,disorders, or conditions, including, but not limited to, cysticfibrosis, emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, protein C deficiency, Type 1hereditary angioedema, lipid processing deficiencies, familialhypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,lysosomal storage diseases, 1-cell disease/pseudo-Hurler,mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, diabetes mellitus, laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetesinsipidus, neurophysiol, nephrogenic, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases, Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, polyglutamine neurologicaldisorders, Huntington, spinocerebullar ataxia type I, spinal and bulbarmuscular atrophy, dentatorubal pallidoluysian, myotonic dystrophy,spongiform encephalopathies, hereditary Creutzfeldt-Jakob disease, Fabrydisease, Straussler-Scheinker syndrome, COPD, dry-eye disease, andSjögren's disease.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing,e.g. activity, by a measurable amount. Compounds that modulate ABCTransporter activity, such as CFTR activity, by increasing the activityof the ABC Transporter, e.g., a CFTR anion channel, are called agonists.Compounds that modulate ABC Transporter activity, such as CFTR activity,by decreasing the activity of the ABC Transporter, e.g., CFTR anionchannel, are called antagonists. An agonist interacts with an ABCTransporter, such as CFTR anion channel, to increase the ability of thereceptor to transduce an intracellular signal in response to endogenousligand binding. An antagonist interacts with an ABC Transporter, such asCFTR, and competes with the endogenous ligand(s) or substrate(s) forbinding site(s) on the receptor to decrease the ability of the receptorto transduce an intracellular signal in response to endogenous ligandbinding.

The phrase “treating or reducing the severity of an ABC Transportermediated disease” refers both to treatments for diseases that aredirectly caused by ABC Transporter and/or CFTR activities andalleviation of symptoms of diseases not directly caused by ABCTransporter and/or CFTR anion channel activities. Examples of diseaseswhose symptoms may be affected by ABC Transporter and/or CFTR activityinclude, but are not limited to, Cystic fibrosis, emphysema, Hereditaryhemochromatosis, Coagulation-Fibrinolysis deficiencies, such as ProteinC deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism,Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma,Glycanosis CDG type 1, emphysema, Congenital hyperthyroidism,Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), Neurophysiol DI, Nephrogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders such as Huntington,Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,Dentatorubal pallidoluysian, and Myotonic dystrophy, as well asSpongiform encephalopathies, such as Hereditary Creutzfeldt-Jakobdisease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eyedisease, and Sjögren's disease.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausolito: 1999, and “March'sAdvanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J.,John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

As used herein the term “aliphatic” encompasses the terms alkyl,alkenyl, alkynyl, each of which being optionally substituted as setforth below.

As used herein, an “alkyl” group refers to a saturated aliphatichydrocarbon group containing 1-12 (e.g., 1-8, 1-6, or 1-4) carbon atoms.An alkyl group can be straight or branched. Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or2-ethylhexyl. An alkyl group can be substituted (i.e., optionallysubstituted) with one or more substituents such as halo, phospho,cycloaliphatic [e.g., cycloalkyl or cycloalkenyl], heterocycloaliphatic[e.g., heterocycloalkyl or heterocycloalkenyl], aryl, heteroaryl,alkoxy, aroyl, heteroaroyl, acyl [e.g., (aliphatic)carbonyl,(cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl], nitro,cyano, amido [e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino,aralkylcarbonylamino, (heterocycloalkyl)carbonylamino,(heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino,heteroaralkylcarbonylamino alkylaminocarbonyl, cycloalkylaminocarbonyl,heterocycloalkylaminocarbonyl, arylaminocarbonyl, orheteroarylaminocarbonyl], amino [e.g., aliphaticamino,cycloaliphaticamino, or heterocycloaliphaticamino], sulfonyl [e.g.,aliphatic-SO₂—], sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl,sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy,heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy,heteroarylalkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy. Withoutlimitation, some examples of substituted alkyls include carboxyalkyl(such as HOOC-alkyl, alkoxycarbonylalkyl, and alkylcarbonyloxyalkyl),cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, aralkyl,(alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as(alkyl-SO₂-amino)alkyl), aminoalkyl, amidoalkyl, (cycloaliphatic)alkyl,or haloalkyl.

As used herein, an “alkenyl” group refers to an aliphatic carbon groupthat contains 2-8 (e.g., 2-12, 2-6, or 2-4) carbon atoms and at leastone double bond. Like an alkyl group, an alkenyl group can be straightor branched. Examples of an alkenyl group include, but are not limitedto allyl, isoprenyl, 2-butenyl, and 2-hexenyl. An alkenyl group can beoptionally substituted with one or more substituents such as halo,phospho, cycloaliphatic [e.g., cycloalkyl or cycloalkenyl],heterocycloaliphatic [e.g., heterocycloalkyl or heterocycloalkenyl],aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, acyl [e.g.,(aliphatic)carbonyl, (cycloaliphatic)carbonyl, or(heterocycloaliphatic)carbonyl], nitro, cyano, amido [e.g.,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino alkylaminocarbonyl,cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl,arylaminocarbonyl, or heteroarylaminocarbonyl], amino [e.g.,aliphaticamino, cycloaliphaticamino, heterocycloaliphaticamino, oraliphaticsulfonylamino], sulfonyl [e.g., alkyl-SO₂—,cycloaliphatic-SO₂—, or aryl-SO₂—], sulfinyl, sulfanyl, sulfoxy, urea,thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl,cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy,aralkyloxy, heteroaralkoxy, alkoxycarbonyl, alkylcarbonyloxy, orhydroxy. Without limitation, some examples of substituted alkenylsinclude cyanoalkenyl, alkoxyalkenyl, acylalkenyl, hydroxyalkenyl,aralkenyl, (alkoxyaryl)alkenyl, (sulfonylamino)alkenyl (such as(alkyl-SO₂-amino)alkenyl), aminoalkenyl, amidoalkenyl,(cycloaliphatic)alkenyl, or haloalkenyl.

As used herein, an “alkynyl” group refers to an aliphatic carbon groupthat contains 2-8 (e.g., 2-12, 2-6, or 2-4) carbon atoms and has atleast one triple bond. An alkynyl group can be straight or branched.Examples of an alkynyl group include, but are not limited to, propargyland butynyl. An alkynyl group can be optionally substituted with one ormore substituents such as aroyl, heteroaroyl, alkoxy, cycloalkyloxy,heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, nitro, carboxy,cyano, halo, hydroxy, sulfo, mercapto, sulfanyl [e.g., aliphaticsulfanylor cycloaliphaticsulfanyl], sulfinyl [e.g., aliphaticsulfinyl orcycloaliphaticsulfinyl], sulfonyl [e.g., aliphatic-SO₂—,aliphaticamino-SO₂—, or cycloaliphatic-SO₂—], amido [e.g.,aminocarbonyl, alkylaminocarbonyl, alkylcarbonylamino,cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl,cycloalkylcarbonylamino, arylaminocarbonyl, arylcarbonylamino,aralkylcarbonylamino, (heterocycloalkyl)carbonylamino,(cycloalkylalkyl)carbonylamino, heteroaralkylcarbonylamino,heteroarylcarbonylamino or heteroarylaminocarbonyl], urea, thiourea,sulfamoyl, sulfamide, alkoxycarbonyl, alkylcarbonyloxy, cycloaliphatic,heterocycloaliphatic, aryl, heteroaryl, acyl [e.g.,(cycloaliphatic)carbonyl or (heterocycloaliphatic)carbonyl], amino[e.g., aliphaticamino], sulfoxy, oxo, carboxy, carbamoyl,(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, or (heteroaryl)alkoxy.

As used herein, an “amido” encompasses both “aminocarbonyl” and“carbonylamino”. These terms when used alone or in connection withanother group refer to an amido group such as —N(R^(X))—C(O)—R^(Y) or—C(O)—N(R^(X))₂, when used terminally, and —C(O)—N(R^(X))— or—N(R^(X))—C(O)—when used internally, wherein R^(X) and R^(Y) are definedbelow. Examples of amido groups include alkylamido (such asalkylcarbonylamino or alkylaminocarbonyl), (heterocycloaliphatic)amido,(heteroaralkyl)amido, (heteroaryl)amido, (heterocycloalkyll)alkylamido,arylamido, aralkylamido, (cycloalkyl)alkylamido, or cycloalkylamido.

As used herein, an “amino” group refers to —NR^(X)R^(Y) wherein each ofR^(X) and R^(Y) is independently hydrogen, aliphatic, cycloaliphatic,(cycloaliphatic)aliphatic, aryl, araliphatic, heterocycloaliphatic,(heterocycloaliphatic)aliphatic, heteroaryl, carboxy, sulfanyl,sulfinyl, sulfonyl, (aliphatic)carbonyl, (cycloaliphatic)carbonyl,((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or(heteroaraliphatic)carbonyl, each of which being defined herein andbeing optionally substituted. Examples of amino groups includealkylamino, dialkylamino, or arylamino. When the term “amino” is not theterminal group (e.g., alkylcarbonylamino), it is represented by—NR^(X)—. R^(X) has the same meaning as defined above.

As used herein, an “aryl” group used alone or as part of a larger moietyas in “aralkyl”, “aralkoxy”, or “aryloxyalkyl” refers to monocyclic(e.g., phenyl); bicyclic (e.g., indenyl, naphthalenyl,tetrahydronaphthyl, tetrahydroindenyl); and tricyclic (e.g., fluorenyltetrahydrofluorenyl, or tetrahydroanthracenyl, anthracenyl) ring systemsin which the monocyclic ring system is aromatic or at least one of therings in a bicyclic or tricyclic ring system is aromatic. The bicyclicand tricyclic groups include benzofused 2-3 membered carbocyclic rings.For example, a benzofused group includes phenyl fused with two or moreC₄₋₈ carbocyclic moieties. An aryl is optionally substituted with one ormore substituents including aliphatic [e.g., alkyl, alkenyl, oralkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic;heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl;alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy;heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl;heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring of abenzofused bicyclic or tricyclic aryl); nitro; carboxy; amido; acyl[e.g., (aliphatic)carbonyl; (cycloaliphatic)carbonyl;((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl;(heterocycloaliphatic)carbonyl;((heterocycloaliphatic)aliphatic)carbonyl; or(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphatic-SO₂— oramino-SO₂—]; sulfinyl [e.g., aliphatic-S(O)— or cycloaliphatic-S(O)—];sulfanyl [e.g., aliphatic-S—]; cyano; halo; hydroxy; mercapto; sulfoxy;urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, anaryl can be unsubstituted.

Non-limiting examples of substituted aryls include haloaryl [e.g.,mono-, di (such as p,m-dihaloaryl), and (trihalo)aryl]; (carboxy)aryl[e.g., (alkoxycarbonyl)aryl, ((aralkyl)carbonyloxy)aryl, and(alkoxycarbonyl)aryl]; (amido)aryl [e.g., (aminocarbonyl)aryl,(((alkylamino)alkyl)aminocarbonyl)aryl, (alkylcarbonyl)aminoaryl,(arylaminocarbonyl)aryl, and (((heteroaryl)amino)carbonyl)aryl];aminoaryl [e.g., ((alkylsulfonyl)amino)aryl or ((dialkyl)amino)aryl];(cyanoalkyl)aryl; (alkoxy)aryl; (sulfamoyl)aryl [e.g.,(aminosulfonyl)aryl]; (alkylsulfonyl)aryl; (cyano)aryl;(hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl; (hydroxy)aryl,((carboxy)alkyl)aryl; (((dialkyl)amino)alkyl)aryl; (nitroalkyl)aryl;(((alkylsulfonyl)amino)alkyl)aryl; ((heterocycloaliphatic)carbonyl)aryl;((alkylsulfonyl)alkyl)aryl; (cyanoalkyl)aryl; (hydroxyalkyl)aryl;(alkylcarbonyl)aryl; alkylaryl; (trihaloalkyl)aryl;p-amino-m-alkoxycarbonylaryl; p-amino-m-cyanoaryl; p-halo-m-aminoaryl;or (m-(heterocycloaliphatic)-o-(alkyl))aryl.

As used herein, an “araliphatic” such as an “aralkyl” group refers to analiphatic group (e.g., a C₁₋₄ alkyl group) that is substituted with anaryl group. “Aliphatic,” “alkyl,” and “aryl” are defined herein. Anexample of an araliphatic such as an aralkyl group is benzyl.

As used herein, an “aralkyl” group refers to an alkyl group (e.g., aC₁₋₄ alkyl group) that is substituted with an aryl group. Both “alkyl”and “aryl” have been defined above. An example of an aralkyl group isbenzyl. An aralkyl is optionally substituted with one or moresubstituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl,including carboxyalkyl, hydroxyalkyl, or haloalkyl such astrifluoromethyl], cycloaliphatic [e.g., cycloalkyl or cycloalkenyl],(cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyll)alkyl, aryl,heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy,heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro,carboxy, alkoxycarbonyl, alkylcarbonyloxy, amido [e.g., aminocarbonyl,alkylcarbonylamino, cycloalkylcarbonylamino,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, or heteroaralkylcarbonylamino], cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, a “bicyclic ring system” includes 8-12 (e.g., 9, 10, or11) membered structures that form two rings, wherein the two rings haveat least one atom in common (e.g., 2 atoms in common). Bicyclic ringsystems include bicycloaliphatics (e.g., bicycloalkyl orbicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclicheteroaryls.

As used herein, a “carbocycle” or “cycloaliphatic” group encompasses a“cycloalkyl” group and a “cycloalkenyl” group, each of which beingoptionally substituted as set forth below.

As used herein, a “cycloalkyl” group refers to a saturated carbocyclicmono- or bicyclic (fused or bridged) ring of 3-10 (e.g., 5-10) carbonatoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, cubyl,octahydro-indenyl, decahydro-naphthyl, bicyclo[3.2.1]octyl,bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decyl,bicyclo[2.2.2]octyl, adamantyl, or((aminocarbonyl)cycloalkyl)cycloalkyl.

A “cycloalkenyl” group, as used herein, refers to a non-aromaticcarbocyclic ring of 3-10 (e.g., 4-8) carbon atoms having one or moredouble bonds. Examples of cycloalkenyl groups include cyclopentenyl,1,4-cyclohexa-di-enyl, cycloheptenyl, cyclooctenyl, hexahydro-indenyl,octahydro-naphthyl, cyclohexenyl, cyclopentenyl, bicyclo[2.2.2]octenyl,or bicyclo[3.3.1]nonenyl.

A cycloalkyl or cycloalkenyl group can be optionally substituted withone or more substituents such as phosphor, aliphatic [e.g., alkyl,alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic) aliphatic,heterocycloaliphatic, (heterocycloaliphatic) aliphatic, aryl,heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy,aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,(cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino,(aryl)carbonylamino, (araliphatic)carbonylamino,(heterocycloaliphatic)carbonylamino,((heterocycloaliphatic)aliphatic)carbonylamino,(heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylaminot nitro,carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g.,(cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, or(heteroaraliphatic)carbonyl], cyano, halo, hydroxy, mercapto, sulfonyl[e.g., alkyl-SO₂— and aryl-SO₂—], sulfinyl [e.g., alkyl-S(O)—], sulfanyl[e.g., alkyl-S—], sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, orcarbamoyl.

As used herein, the term “heterocycle” or “heterocycloaliphatic”encompasses a heterocycloalkyl group and a heterocycloalkenyl group,each of which being optionally substituted as set forth below.

As used herein, a “heterocycloalkyl” group refers to a 3-10 memberedmono- or bicylic (fused or bridged) (e.g., 5- to 10-membered mono- orbicyclic) saturated ring structure, in which one or more of the ringatoms is a heteroatom (e.g., N, O, S, or combinations thereof). Examplesof a heterocycloalkyl group include piperidyl, piperazyl,tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl,1,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl,octahydrobenzofuryl, octahydrochromenyl, octahydrothiochromenyl,octahydroindolyl, octahydropyrindinyl, decahydroquinolinyl,octahydrobenzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl,1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, and2,6-dioxa-tricyclo[3.3.1.0^(3,7)]nonyl. A monocyclic heterocycloalkylgroup can be fused with a phenyl moiety to form structures, such astetrahydroisoquinoline, which would be categorized as heteroaryls.

A “heterocycloalkenyl” group, as used herein, refers to a mono- orbicylic (e.g., 5- to 10-membered mono- or bicyclic) non-aromatic ringstructure having one or more double bonds, and wherein one or more ofthe ring atoms is a heteroatom (e.g., N, O, or S). Monocyclic andbicyclic heterocycloaliphatics are numbered according to standardchemical nomenclature.

A heterocycloalkyl or heterocycloalkenyl group can be optionallysubstituted with one or more substituents such as phosphor, aliphatic[e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic,(cycloaliphatic)aliphatic, heterocycloaliphatic,(heterocycloaliphatic)aliphatic, aryl, heteroaryl, alkoxy,(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy,(araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl, amino,amido [e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino,((cycloaliphatic) aliphatic)carbonylamino, (aryl)carbonylamino,(araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino,((heterocycloaliphatic) aliphatic)carbonylamino,(heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro,carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g.,(cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, or(heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy, mercapto,sulfonyl [e.g., alkylsulfonyl or arylsulfonyl], sulfinyl [e.g.,alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

A “heteroaryl” group, as used herein, refers to a monocyclic, bicyclic,or tricyclic ring system having 4 to 15 ring atoms wherein one or moreof the ring atoms is a heteroatom (e.g., N, O, S, or combinationsthereof) and in which the monocyclic ring system is aromatic or at leastone of the rings in the bicyclic or tricyclic ring systems is aromatic.A heteroaryl group includes a benzofused ring system having 2 to 3rings. For example, a benzofused group includes benzo fused with one ortwo 4 to 8 membered heterocycloaliphatic moieties (e.g., indolizyl,indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl,benzo[b]thiophenyl, quinolinyl, or isoquinolinyl). Some examples ofheteroaryl are azetidinyl, pyridyl, 1H-indazolyl, furyl, pyrrolyl,thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl,isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine,dihydroindole, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thiophenyl,indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl,quinazolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl,4H-quinolizyl, benzo-1,2,5-thiadiazolyl, or 1,8-naphthyridyl.

Without limitation, monocyclic heteroaryls include furyl, thiophenyl,2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl, pyrazolyl,isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pranyl,pyridyl, pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl.Monocyclic heteroaryls are numbered according to standard chemicalnomenclature.

Without limitation, bicyclic heteroaryls include indolizyl, indolyl,isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl,quinolinyl, isoquinolinyl, indolizinyl, isoindolyl, indolyl,benzo[b]furyl, bexo[b]thiophenyl, indazolyl, benzimidazyl,benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl,phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl.Bicyclic heteroaryls are numbered according to standard chemicalnomenclature.

A heteroaryl is optionally substituted with one or more substituentssuch as aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic;(cycloaliphatic)aliphatic; heterocycloaliphatic;(heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy;(cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy;(araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo(on a non-aromatic carbocyclic or heterocyclic ring of a bicyclic ortricyclic heteroaryl); carboxy; amido; acyl [e.g., aliphaticcarbonyl;(cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl;(araliphatic)carbonyl; (heterocycloaliphatic)carbonyl;((heterocycloaliphatic)aliphatic)carbonyl; or(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl oraminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g.,aliphaticsulfanyl]; nitro; cyano; halo; hydroxy; mercapto; sulfoxy;urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, aheteroaryl can be unsubstituted.

Non-limiting examples of substituted heteroaryls include(halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl];(carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl]; cyanoheteroaryl;aminoheteroaryl [e.g., ((alkylsulfonyl)amino)heteroaryl and((dialkyl)amino)heteroaryl]; (amido)heteroaryl [e.g.,aminocarbonylheteroaryl, ((alkylcarbonyl)amino)heteroaryl,((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl,(((heteroaryl)amino)carbonyl)heteroaryl,((heterocycloaliphatic)carbonyl)heteroaryl, and((alkylcarbonyl)amino)heteroaryl]; (cyanoalkyl)heteroaryl;(alkoxy)heteroaryl; (sulfamoyl)heteroaryl [e.g.,(aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g.,(alkylsulfonyl)heteroaryl]; (hydroxyalkyl)heteroaryl;(alkoxyalkyl)heteroaryl; (hydroxy)heteroaryl;((carboxy)alkyl)heteroaryl; (((dialkyl)amino)alkyl]heteroaryl;(heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl;(nitroalkyl)heteroaryl; (((alkylsulfonyl)amino)alkyl)heteroaryl;((alkylsulfonyl)alkyl)heteroaryl; (cyanoalkyl)heteroaryl;(acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl]; (alkyl)heteroaryl,and (haloalkyl)heteroaryl [e.g., trihaloalkylheteroaryl].

A “heteroaraliphatic” (such as a heteroaralkyl group) as used herein,refers to an aliphatic group (e.g., a C₁₋₄ alkyl group) that issubstituted with a heteroaryl group. “Aliphatic,” “alkyl,” and“heteroaryl” have been defined above.

A “heteroaralkyl” group, as used herein, refers to an alkyl group (e.g.,a C₁₋₄ alkyl group) that is substituted with a heteroaryl group. Both“alkyl” and “heteroaryl” have been defined above. A heteroaralkyl isoptionally substituted with one or more substituents such as alkyl(including carboxyalkyl, hydroxyalkyl, and haloalkyl such astrifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl,heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy,cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy,heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl,alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino,cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino,arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, “cyclic moiety” and “cyclic group” refer to mono-, bi-,and tri-cyclic ring systems including cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl, each of which has beenpreviously defined.

As used herein, a “bridged bicyclic ring system” refers to a bicyclicheterocyclicaliphatic ring system or bicyclic cycloaliphatic ring systemin which the rings are bridged. Examples of bridged bicyclic ringsystems include, but are not limited to, adamantanyl, norbornanyl,bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl,bicyclo[3.2.3]nonyl, 2-oxabicyclo[2.2.2]octyl, 1-azabicyclo[2.2.2]octyl,3-azabicyclo[3.2.1]octyl, and 2,6-dioxa-tricyclo[3.3.1.0^(3,7)]nonyl. Abridged bicyclic ring system can be optionally substituted with one ormore substituents such as alkyl (including carboxyalkyl, hydroxyalkyl,and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl,(cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl,heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy,heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro,carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl,alkylcarbonylamino, cycloalkylcarbonylamino,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, an “acyl” group refers to a formyl group or R^(X)—C(O)—(such as alkyl-C(O)—, also referred to as “alkylcarbonyl”) where Rx and“alkyl” have been defined previously. Acetyl and pivaloyl are examplesof acyl groups.

As used herein, an “aroyl” or “heteroaroyl” refers to an aryl-C(O)— or aheteroaryl-C(O)—. The aryl and heteroaryl portion of the aroyl orheteroaroyl is optionally substituted as previously defined.

As used herein, an “alkoxy” group refers to an alkyl-O— group where“alkyl” has been defined previously.

As used herein, a “carbamoyl” group refers to a group having thestructure —O—CO—NR^(X)R^(Y) or —NR^(X)—CO—O—R^(Z), wherein R^(X) andR^(Y) have been defined above and R^(Z) can be aliphatic, aryl,araliphatic, heterocycloaliphatic, heteroaryl, or heteroaraliphatic.

As used herein, a “carboxy” group refers to —COOH, —COOR^(X), —OC(O)H,—OC(O)R^(X), when used as a terminal group; or —OC(O)— or —C(O)O— whenused as an internal group.

As used herein, a “haloaliphatic” group refers to an aliphatic groupsubstituted with 1-3 halogen. For instance, the term haloalkyl includesthe group —CF₃.

As used herein, a “mercapto” group refers to —SH.

As used herein, a “sulfo” group refers to —SO₃H or —SO₃Rx when usedterminally or —S(O)₃— when used internally.

As used herein, a “sulfamide” group refers to the structure—NR^(X)—S(O)₂—NR^(Y)R^(Z) when used terminally and —NR^(X)—S(O)₂—NR^(Y)—when used internally, wherein R^(X), R^(Y), and R^(Z) have been definedabove.

As used herein, a “sulfonamide” group refers to the structure—S(O)₂—NR^(X)R^(Y) or —NR^(X)—S(O)₂—R^(Z) when used terminally; or—S(O)₂—NR^(X)— or —NR^(X)—S(O)₂— when used internally, wherein R^(X),R^(Y), and R^(Z) are defined above.

As used herein a “sulfanyl” group refers to —S—R^(X) when usedterminally and —S— when used internally, wherein R^(X) has been definedabove. Examples of sulfanyls include aliphatic-S—, cycloaliphatic-S—,aryl-S—, or the like.

As used herein a “sulfinyl” group refers to —S(O)—R^(X) when usedterminally and —S(O)—when used internally, wherein R^(X) has beendefined above. Exemplary sulfinyl groups include aliphatic-S(O)—,aryl-S(O)—, (cycloaliphatic(aliphatic))-S(O)—, cycloalkyl-S(O)—,heterocycloaliphatic-S(O)—, heteroaryl-S(O)—, or the like.

As used herein, a “sulfonyl” group refers to —S(O)₂—R^(X) when usedterminally and —S(O)₂— when used internally, wherein R^(X) has beendefined above. Exemplary sulfonyl groups include aliphatic-S(O)₂—,aryl-S(O)₂—, (cycloaliphatic(aliphatic))-S(O)₂—, cycloaliphatic-S(O)₂—,heterocycloaliphatic-S(O)₂—, heteroaryl-S(O)₂—,(cycloaliphatic(amido(aliphatic)))-S(O)₂— or the like.

As used herein, a “sulfoxy” group refers to —O—SO—Rx or —SO—O—Rx, whenused terminally and —O—S(O)— or —S(O)—O— when used internally, where Rxhas been defined above.

As used herein, a “halogen” or “halo” group refers to fluorine,chlorine, bromine or iodine.

As used herein, an “alkoxycarbonyl,” which is encompassed by the termcarboxy, used alone or in connection with another group refers to agroup such as alkyl-O—C(O)—.

As used herein, an “alkoxyalkyl” refers to an alkyl group such asalkyl-O-alkyl-, wherein alkyl has been defined above.

As used herein, a “carbonyl” refers to —C(O)—.

As used herein, an “oxo” refers to ═O.

As used herein, the term “phospho” refers to phosphinates andphosphonates. Examples of phosphinates and phosphonates include—P(O)(R^(P))₂, wherein R^(P) is aliphatic, alkoxy, aryloxy,heteroaryloxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy aryl,heteroaryl, cycloaliphatic or amino.

As used herein, an “aminoalkyl” refers to the structure(R^(X))₂N-alkyl-.

As used herein, a “cyanoalkyl” refers to the structure (NC)-alkyl-.

As used herein, a “urea” group refers to the structure—NR^(X)—CO—NR^(Y)R^(Z) and a “thiourea” group refers to the structure—NR^(X)—CS—NR^(Y)R^(Z) when used terminally and —NR^(X)—CO—NR^(Y)— or—NR^(X)—CS—NR^(Y)— when used internally, wherein R^(X), R^(Y), and R^(Z)have been defined above.

As used herein, a “guanidine” group refers to the structure—N═C(N(R^(X)R^(Y)))N(R^(X)R^(Y)) or

—NR^(X)—C(═NR^(X))NR^(X)R^(Y) wherein R^(X) and R^(Y) have been definedabove.

As used herein, the term “amidino” group refers to the structure—C═(NR^(X))N(R^(X)R^(Y)) wherein R^(X) and R^(Y) have been definedabove.

In general, the term “vicinal” refers to the placement of substituentson a group that includes two or more carbon atoms, wherein thesubstituents are attached to adjacent carbon atoms.

In general, the term “geminal” refers to the placement of substituentson a group that includes two or more carbon atoms, wherein thesubstituents are attached to the same carbon atom.

The terms “terminally” and “internally” refer to the location of a groupwithin a substituent. A group is terminal when the group is present atthe end of the substituent not further bonded to the rest of thechemical structure. Carboxyalkyl, i.e., R^(X)O(O)C-alkyl is an exampleof a carboxy group used terminally. A group is internal when the groupis present in the middle of a substituent of the chemical structure.Alkylcarboxy (e.g., alkyl-C(O)O— or alkyl-OC(O)—) and alkylcarboxyaryl(e.g., alkyl-C(O)O-aryl- or alkyl-O(CO)-aryl-) are examples of carboxygroups used internally.

As used herein, an “aliphatic chain” refers to a branched or straightaliphatic group (e.g., alkyl groups, alkenyl groups, or alkynyl groups).A straight aliphatic chain has the structure —[CH₂]—, where v is 1-12. Abranched aliphatic chain is a straight aliphatic chain that issubstituted with one or more aliphatic groups. A branched aliphaticchain has the structure —[CQQ]_(v)— where each Q is independently ahydrogen or an aliphatic group; however, Q shall be an aliphatic groupin at least one instance. The term aliphatic chain includes alkylchains, alkenyl chains, and alkynyl chains, where alkyl, alkenyl, andalkynyl are defined above.

The phrase “optionally substituted” is used interchangeably with thephrase “substituted or unsubstituted.” As described herein, compounds ofthe invention can optionally be substituted with one or moresubstituents, such as are illustrated generally above, or as exemplifiedby particular classes, subclasses, and species of the invention. Asdescribed herein, the variables R₁, R₂, and R₃, and other variablescontained in formulae described herein encompass specific groups, suchas alkyl. Unless otherwise noted, each of the specific groups for thevariables R₁, R₂, and R₃, and other variables contained therein can beoptionally substituted with one or more substituents described herein.Each substituent of a specific group is further optionally substitutedwith one to three of halo, cyano, oxo, alkoxy, hydroxy, amino, nitro,aryl, cycloaliphatic, heterocycloaliphatic, heteroaryl, haloalkyl, andalkyl. For instance, an alkyl group can be substituted withalkylsulfanyl and the alkylsulfanyl can be optionally substituted withone to three of halo, cyano, oxo, alkoxy, hydroxy, amino, nitro, aryl,haloalkyl, and alkyl. As an additional example, the cycloalkyl portionof a (cycloalkyl)carbonylamino can be optionally substituted with one tothree of halo, cyano, alkoxy, hydroxy, nitro, haloalkyl, and alkyl. Whentwo alkoxy groups are bound to the same atom or adjacent atoms, the twoalkoxy groups can form a ring together with the atom(s) to which theyare bound.

In general, the term “substituted,” whether preceded by the term“optionally” or not, refers to the replacement of hydrogen radicals in agiven structure with the radical of a specified substituent. Specificsubstituents are described above in the definitions and below in thedescription of compounds and examples thereof. Unless otherwiseindicated, an optionally substituted group can have a substituent ateach substitutable position of the group, and when more than oneposition in any given structure can be substituted with more than onesubstituent selected from a specified group, the substituent can beeither the same or different at every position. A ring substituent, suchas a heterocycloalkyl, can be bound to another ring, such as acycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings shareone common atom. As one of ordinary skill in the art will recognize,combinations of substituents envisioned by this invention are thosecombinations that result in the formation of stable or chemicallyfeasible compounds.

The phrase “stable or chemically feasible,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and preferablytheir recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

As used herein, an “effective amount” is defined as the amount requiredto confer a therapeutic effect on the treated patient, and is typicallydetermined based on age, surface area, weight, and condition of thepatient. The interrelationship of dosages for animals and humans (basedon milligrams per meter squared of body surface) is described byFreireich et al., Cancer Chemother. Rep., 50: 219 (1966). Body surfacearea may be approximately determined from height and weight of thepatient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley,N.Y., 537 (1970). As used herein, “patient” refers to a mammal,including a human.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. Additionally, unless otherwise stated, structuresdepicted herein are also meant to include compounds that differ only inthe presence of one or more isotopically enriched atoms. For example,compounds having the present structures except for the replacement ofhydrogen by deuterium or tritium, or the replacement of a carbon by a¹³C— or ¹⁴C-enriched carbon are within the scope of this invention. Suchcompounds are useful, for example, as analytical tools or probes inbiological assays, or as therapeutic agents.

Compounds of the present invention are useful modulators of ABCtransporters and are useful in the treatment of ABC transporter mediateddiseases.

In another embodiment, the invention features a compound of formula I,wherein two R taken together form —OCF₂O—, R₁ is H, and R₂ is F. Inanother embodiment, two R taken together form —OCF₂O—, R₁ is H, R₂ is F,and R₃ is CH₃. In another embodiment, two R taken together form —OCF₂O—,R₁ is H, R₂ is F, R₃ is CH₃, and X is CO₂H. In another embodiment, two Rtaken together form —OCF₂O—, R₁ is H, R₂ is F, R₃ is CH₃, X is CO₂H, andY is OH.

In another embodiment, the invention features a compound of formula II,wherein two R taken together form —OCF₂O—, R₁ is H, and R₂ is F. Inanother embodiment, two R taken together form —OCF₂O—, R₁ is H, R₂ is F,and R₃ is CH₃.

In another embodiment, the invention features a compound having formulaIa:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   R₂ is H or halo.

In another embodiment, R₂ is F.

In another embodiment, the invention features a compound having formulaIIa:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   R₂ is H or halo.

In another embodiment, R₂ is F.

In another embodiment, the invention features the compound

In another embodiment, the invention features the compound

In another aspect, the present invention features a pharmaceuticalcomposition comprising (i) a compound according to any one of claims 1to 12; and (ii) a pharmaceutically acceptable carrier. In anotherembodiment, the composition further comprises an additional agentselected from a mucolytic agent, bronchodialator, an anti-biotic, ananti-infective agent, an anti-inflammatory agent, CFTR corrector, CFTRpotentiator, or a nutritional agent.

In another aspect, the present invention features a method of increasingthe number of functional ABC transporters in a membrane of a cell,comprising the step of contacting the cell with a compound of theinvention. In another embodiment, the ABC transporter is CFTR.

In another aspect, the present invention features a method of treating acondition, disease, or disorder in a subject implicated by ABCtransporter activity, comprising the step of administering to thesubject a compound or composition of the invention.

In another embodiment, the condition, disease, or disorder is selectedfrom cystic fibrosis, emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, protein C deficiency, Type 1hereditary angioedema, lipid processing deficiencies, familialhypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,lysosomal storage diseases, I-cell disease/pseudo-Hurler,mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, diabetes mellitus, laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetesinsipidus (di), neurophyseal di, neprogenic DI, Charcot-Marie Toothsyndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, polyglutamineneurological disorders, Huntington, spinocerebullar ataxia type I,spinal and bulbar muscular atrophy, dentatorubal pallidoluysian,myotonic dystrophy, spongiform encephalopathies, hereditaryCreutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome,COPD, dry-eye disease, or Sjögren's disease. In another embodiment, thecondition, disease, or disorder is selected from cystic fibrosis,emphysema, COPD, or dry-eye disease.

In another aspect the present invention features a kit for use inmeasuring the activity of a ABC transporter or a fragment thereof in abiological sample in vitro or in vivo, comprising: (i) a compound of theinvention; and (ii) instructions for: a) contacting the compound withthe biological sample; and b) measuring activity of said ABC transporteror a fragment thereof.

In another embodiment, the kit further comprises instructions for a)contacting an additional compound with the biological sample; b)measuring the activity of said ABC transporter or a fragment thereof inthe presence of said additional compound, and c) comparing the activityof the ABC transporter in the presence of the additional compound withthe density of the ABC transporter in the presence of the firstcompound.

In another aspect, the invention comprises a process for preparing acompound of formula Ia

wherein the variables are as described above, comprising treatment of acompound of formula I-2 with a base.

In one embodiment of this aspect, R₂ is H or F.

In another embodiment, treatment comprises contacting the compound offormula I-2 with a base in the presence of a solvent. In one embodiment,the base is an alkali or alkali metal hydroxide or carbonate. In oneembodiment, the base is selected from Na₂CO₃, NaHCO₃, NaOH and LiOH.Typically a stoichiometric excess of the base is used. Typically fromabout 2 to about 10 equivalents of the base are used relative to themoles of the compound of formula I-2. More typically, about 4 to about 6molar equivalents of the base are used.

In one embodiment, the solvent is a polar solvent, such as an alcohol oran ether, that is used alone or that is admixed with another liquid. Inone embodiment, the solvent is methanol.

In another embodiment, the solvent is methanol admixed withacetonitrile. In another embodiment, the solvent is methanol admixedwith isopropanol. Typically about 4 to about 8 volumes of solvent areused. More typically, about 5 to about 7 volumes of solvent are used.

The conversion of the compound of formula I-2 to Ia is typicallyperformed at a sufficient temperature for a sufficient time to allow forconversion of the starting material to the product. Typically, thetemperature is approximately room temperature.

In another embodiment, the process for preparing a compound of formulaIa from a compound of formula I-2 comprises contacting the compound offormula I-2 with an alkali or alkali earth metal base which is ahydroxide or carbonate in the presence of a solvent. In one embodiment,the alkali or alkali earth metal base is Na₂CO₃ and the solvent ismethanol.

In another aspect, the invention comprises a process for preparing acompound of formula I-2 from a compound of formula I-3

comprising contacting the compound of formula I-3 with an oxidant in thepresence of a solvent to provide a compound of formula I-2; wherein thevariables are as described above.

In one embodiment of this aspect, R₂ is H or F.

In one embodiment, the oxidant is selected from the group consisting ofKMnO₄ and NaMnO₄. In one embodiment, the oxidant is NaMnO₄. Typically, amolar excess of the oxidant is used relative the moles of the compoundof formula I-3. Typically, 1.01 to 1.2 molar equivalents of oxidant areused. More typically, 1.05 equivalents of the oxidant are used.

In one embodiment, the solvent is a polar aprotic solvent that is usedalone or that is admixed with another liquid. In one embodiment, thesolvent is acetone. Typically about 5 to about 15 volumes of solvent areused. More typically, about 7 to about 13 volumes of solvent are used,and more typically, about 9 to about 11 volumes of solvent are used.

The conversion of the compound of formula I-3 to 1-2 is typicallyperformed at a sufficient temperature for sufficient time to allow forconversion of the starting material to the product. Typically, thetemperature is below room temperature. For example, the temperature isapproximately −10 to about 10° C. More typically, the temperature isapproximately −5 to about 5° C.

In another embodiment, the process for preparing a compound of formulaI-2 from a compound of formula I-3 comprises contacting the compound offormula I-3 with an oxidant in the presence of a solvent. In oneembodiment, the oxidant is NaMnO₄ and the solvent is acetone.

In another aspect, the invention comprises a process for preparing acompound of formula I-3 from a compound of formula I-4

comprising contacting the compound of formula I-4 with an oxidant in thepresence of a solvent to provide a compound of formula I-3; wherein thevariables are as described above.

In one embodiment of this aspect, R₂ is H or F.

In one embodiment, the oxidant is selected from the group consisting ofsulfur trioxide pyridine complex, pyridinium dichromate (PDC),N-chlorosuccinimide (NCS)/benzenesulfenamide (PhSNHtBu) optionally inthe presence of 2-methyl-2-butene as a chlorine scavenger, RuCl₃/NaIO₄,tetramethylpiperidine N-oxide (TEMPO)/bisacetoxyiodobenzene(BIAB)/NaHCO₃, and 2-iodoxybenzoic acid (IBX). In one embodiment, theoxidant is N-chlorosuccinimide (NCS)/benzenesulfenamide (PhSNHtBu) inthe presence of a tertiary amine base and 2-methyl-2-butene as achlorine scavenger. Tertiary amine bases that can be used in thisprocess are well known to the skilled practitioner and include, forexample, triethyl amine, diisopropylethyl amine, DBU, DBN, andcollidine. In one embodiment, the tertiary amine base is collidine.Typically, a catalytic amount of PhSNHtBu is used, relative to thenumber of moles of the compound of formula I-4, and the NCS, tertiaryamine base, and 2-methyl-2-butene are used in molar excess. For example0.1 to 0.3 molar equivalent of PhSNHtBu is used, relative to the numberof moles of the compound of formula I-4, and 1.1 to 1.5 equivalents ofNCS, 1-3 equivalents of tertiary amine base, and 1-3 molar equivalentsof 2-methyl-2-butene are used. More typically, For example 0.15 to 0.25molar equivalent of PhSNHtBu is used, relative to the number of moles ofthe compound of formula I-4, and 1.1 to 1.3 equivalents of NCS, 1.5-2.5equivalents of tertiary amine base, and 1.5-2.5 molar equivalents of2-methyl-2-butene are used.

In one embodiment, the solvent is a polar aprotic solvent that is usedalone or that is admixed with another liquid. In one embodiment, thesolvent is dichloromethane. Typically about 5 to about 10 volumes ofsolvent are used. More typically, about 6 to about 8 volumes of solventare used.

The conversion of the compound of formula I-4 to 1-3 is typicallyperformed at a sufficient temperature for sufficient time to allow forconversion of the starting material to the product. Typically, thetemperature is below room temperature. For example, the temperature isapproximately −10 to about 10° C. More typically, the temperature isapproximately −5 to about 5° C.

In another embodiment, the process for preparing a compound of formulaI-3 from a compound of formula I-4 comprises contacting the compound offormula I-3 with is N-chlorosuccinimide (NCS)/benzenesulfenamide(PhSNHtBu) in the presence of a tertiary amine base and2-methyl-2-butene as a chlorine scavenger in the presence of a solvent.In one embodiment, the tertiary amine base and the solvent isdichloromethane.

In another aspect, the invention comprises a process for preparing acompound of formula I-4 from a compound of formula I-5

comprising contacting the compound of formula I-4 with carbonyldiimidazole (CDI) in the presence of a solvent to provide a compound offormula I-4; wherein the variables are as described above.

In one embodiment of this aspect, R₂ is H or F.

In one embodiment, a molar excess of CDI is used relative to the molesof the compound of formula I-5. Typically, 1.1 to 3 molar equivalents ofCDI are used. More typically, 1.5 to 2.5 molar equivalents of CDI areused.

In one embodiment, the solvent is a polar solvent that is used alone orthat is admixed with another liquid. In one embodiment, the solvent isan ether or dichloromethane. In one embodiment, the solvent isdichloromethane. Typically about 12 to about 16 volumes of solvent areused. More typically, about 13 to about 15 volumes of solvent are used.

The conversion of the compound of formula I-5 to 1-4 is typicallyperformed at a sufficient temperature for sufficient time to allow forconversion of the starting material to the product. Typically, thetemperature is below room temperature. For example, the temperature isapproximately −20 to about 10° C. More typically, the temperature isapproximately −15 to about 5° C.

In another embodiment, the process for preparing a compound of formulaI-4 from a compound of formula I-5 comprises contacting the compound offormula I-5 with CDI in the presence of a solvent. In one embodiment,the solvent is dichloromethane.

In another aspect, the invention provides a process for preparing acompound of formula Ia

comprising converting an ester of formula I-1 to a compound of formulaIa:

-   -   wherein independently for each occurrence:    -   R₂ is H or halo; and    -   R₄ is C₁-C₆ alkyl or benzyl.

In one embodiment of this aspect, R₂ is H or F, and R₄ is methyl, ethyl,isopropyl, butyl, or benzyl.

In another embodiment, R₂ is H or F, and R₄ is isopropyl or benzyl.

In another embodiment, converting comprises contacting the compound offormula I-1 with a base in the presence of a solvent. In one embodiment,the base is an alkali or alkali metal hydroxide. In one embodiment, thebase is NaOH or LiOH.

In one embodiment, the solvent is a polar solvent, such an alcohol or anether, that is used alone or that is admixed with another liquid. In oneembodiment, the solvent is methanol. In another embodiment, the solventis methanol admixed with water. In another embodiment, the solvent istetrahydrofuran. In another embodiment, the solvent is tetrahydrofuranadmixed with water.

The conversion of the compound of formula I-1 to Ia is typicallyperformed at a temperature for sufficient time to allow for conversionof the starting material to the product. Typically, the temperature isabove room temperature. More typically, the temperature is approximately50° C. Typically reaction times are from about 1 hour to about 24 hours.

In another embodiment, the process for preparing a compound of formulaIa from a compound of formula I-1 comprises contacting the compound offormula I-1 with an alkali or alkali earth metal hydroxide in thepresence of a solvent. In one embodiment, the alkali or alkali earthmetal hydroxide is LiOH or NaOH and the solvent is methanol alone oradmixed with water, or THF alone or admixed with water.

In another aspect, the invention comprises a process for preparing acompound of formula IIa

or a pharmaceutically acceptable salt thereof, wherein R₂ is H or halo;comprising:

-   -   converting the compound of formula I-3 to the compound of        formula IIa.

In one embodiment of this aspect, R₂ is H or F.

In one embodiment, treatment comprises contacting the compound offormula I-3 with a base in the presence of a solvent. In one embodiment,the base is an alkali or alkali metal hydroxide or carbonate. In oneembodiment, the base is selected from NaOH, KOH, and LiOH. In oneembodiment, the base is NaOH. Typically a stoichiometric excess of thebase is used. Typically from about 2 to about 10 equivalents of the baseare used relative to the moles of the compound of formula I-3. Moretypically, about 4 to about 6 molar equivalents of the base are used.Typically, the base is used as a solution in water.

In one embodiment, the solvent is a polar solvent, such as an alcohol oran ether, that is used alone or that is admixed with another liquid. Inone embodiment, the solvent is methanol.

In another embodiment, the solvent is methanol admixed withacetonitrile. In another embodiment, the solvent is methanol admixedwith isopropanol. Typically about 4 to about 8 volumes of solvent areused. More typically, about 5 to about 7 volumes of solvent are used.

The conversion of the compound of formula I-2 to Ia is typicallyperformed at a temperature for sufficient time to allow for conversionof the starting material to the product. Typically, the temperature isapproximately room temperature.

In another embodiment, the process for preparing a compound of formulaIa from a compound of formula I-2 comprises contacting the compound offormula I-2 with an alkali or alkali earth metal base which is ahydroxide or carbonate in the presence of a solvent. In one embodiment,the alkali or alkali earth metal base is Na₂CO₃ and the solvent ismethanol.

In another aspect, the invention comprises a process for preparing acompound of formula Ia

wherein the variables are as described above, comprising:

(a) contacting the compound of formula I-3 with an oxidant in thepresence of a solvent as provided above to give a compound of formulaI-2;

and

(b) contacting the compound of formula I-1 with a base in the presenceof a solvent as provided above to give a compound of formula Ia.

In one embodiment of this aspect, R₂ is H or F.

In another aspect, the invention comprises a process for preparing acompound of formula Ia

wherein the variables are as described above, comprising:

(a) contacting the compound of formula I-4 with an oxidant in thepresence of a solvent as provided above to give a compound of formulaI-3

(b) contacting the compound of formula I-3 with an oxidant in thepresence of a solvent as provided above to give compound of formula I-2;

and

(c) contacting the compound of formula I-1 with a base in the presenceof a solvent as provided above to give a compound of formula Ia.

In one embodiment of this aspect, R₂ is H or F.

In another aspect, the invention comprises a process for preparing acompound of formula Ia

wherein the variables are as described above, comprising:

(a) contacting the compound of formula I-4 with carbonyl diimidazole(CDI) in the presence of a solvent as provided above to give a compoundof formula I-4

(b) contacting the compound of formula I-4 with an oxidant in thepresence of a solvent as provided above to give a compound of formulaI-3

(c) contacting the compound of formula I-3 with an oxidant in thepresence of a solvent as provided above to give compound of formula I-2;

and

(d) contacting the compound of formula I-1 with a base in the presenceof a solvent as provided above to give a compound of formula Ia.

In one embodiment of this aspect, R₂ is H or F.

In another aspect, the invention comprises a process for preparing acompound of formula IIa

wherein the variables are as described above, comprising:

(a) contacting the compound of formula I-4 with an oxidant in thepresence of a solvent as provided above to give a compound of formulaI-3

and

(b) contacting the compound of formula I-3 with a base in the presenceof a solvent as provided above to give a compound of formula IIa.

In one embodiment of this aspect, R₂ is H or F.

In another aspect, the invention comprises a process for preparing acompound of formula IIa

wherein the variables are as described above, comprising:

(a) contacting the compound of formula I-4 with carbonyl diimidazole(CDI) in the presence of a solvent as provided above to give a compoundof formula I-4;

(b) contacting the compound of formula I-4 with an oxidant in thepresence of a solvent as provided above to give a compound of formulaI-3;

and

(c) contacting the compound of formula I-3 with a base in the presenceof a solvent as provided above to give a compound of formula IIa;

In one embodiment of this aspect, R₂ is H or F.

In another aspect, the invention comprises a compound which is:

wherein R₂ and R₄ are defined as above.

In another aspect, the invention comprises a compound which is:

wherein R₂ and R₄ are defined as above.

In another aspect, the invention comprises a compound which is:

wherein R₄ is iPr or benzyl.

In another aspect, the invention comprises a compound which is:

wherein R₄ is iPr or benzyl.

Overview of the Synthesis of Compounds of Formula I and Formula II

Compounds of formula I can be prepared by coupling an acid chloridemoiety with an amine moiety followed by ring closure according tofollowing Schemes 1 to 5.

Scheme 1 depicts the preparation of R and R₁ substitutedbenzo-cyclopropanecarbonyl chloride, which is used in Scheme 3 to makethe amide linkage of compounds of formula I.

Scheme 2 provides an alternative synthesis of the requisite acidchloride. R-substituted 5-bromobenzene is coupled with ethylcyanoacetate in the presence of a palladium catalyst to form thecorresponding alpha cyano ethyl ester. Saponification of the estermoiety to the carboxylic acid gives the cyanoethyl compound. Alkylationof the cyanoethyl compound with R₁ substituted 1-bromo-2-chloro ethanein the presence of base gives the cyanocyclopropyl compound. Treatmentof the cyanocyclopropyl compound with base gives the carboxylate salt,which is converted to the carboxylic acid by treatment with acid.Conversion of the carboxylic acid to the acid chloride is thenaccomplished using a chlorinating agent such as thionyl chloride or thelike.

Scheme 3 provides an overview of the synthesis of the amine moiety ofcompounds of formula I via a Sonagashira/cyclization protocol. From thesilyl protected propargyl alcohol shown, conversion to the propargylchloride followed by formation of the Grignard reagent and subsequentnucleophilic substitution provides((R₃-substituted-but-3-ynyloxy)methyl)benzene, which is used in anotherstep of the synthesis. To complete the amine moiety,4-nitro-3-R₂-aniline is first brominated, and then converted to thetoluenesulfonic acid salt of(R)-1-(4-amino-2-bromo-5-R₂-substituted-phenylamino)-3-(benzyloxy)propan-2-olin a two-step process beginning with alkylation of the aniline aminogroup by (R)-2-(benzyloxymethyl)oxirane, followed by reduction of thenitro group to the corresponding amine. Palladium catalyzed coupling ofthe product with ((R₃-substituted-but-3-ynyloxy)methyl)benzene(discussed above) provides the intermediate akynyl compound which isthen cyclized to the indole moiety to produce the benzyl protected aminemoiety.

Scheme 4 depicts the coupling of the Acid and Amine moieties. In thefirst step, (R)-1-(5-amino-241-(benzyloxy)-2-methylpropan-2-yl)-6-R₂-1H-indol-1-yl)-3-(benzyloxy)propan-2-olis coupled with 1-(R-substituted-5-yl)cyclopropanecarbonyl chloride toprovide the benzyl protected precursors to compounds of formula I. Thisstep can be performed in the presence of a base and a solvent. The basecan be an organic base such as triethylamine, and the solvent can be anorganic solvent such as DCM or a mixture of DCM and toluene.

In the last step, the benzylated intermediate is deprotected to produceprecursors to compounds of formula I. The deprotection step can beaccomplished using reducing conditions sufficient to remove the benzylgroup. The reducing conditions can be hydrogenation conditions such ashydrogen gas in the presence of a palladium catalyst to provide thealcohol. This material can be converted directly to a compound offormula I via microbial oxidation.

Scheme 5 provides the preparation of a compound of formula II. Theproduct depicted in Scheme 4 is oxidized with pyridinium dichromate indichloromethane to provide the compound of formula II.

Scheme 6 provides the preparation of a compound of formula I from acompound of formula II. Oxidation of the compound of formula II depictedin Scheme 5 with silver carbonate in the presence of Celite initiallygives a cyclic lactone product, which is hydrolyzed in the presence of2N sodium hydroxide to provide the compound of formula I.

Scheme 7 provides an alternative process for preparing a compound offormula I. The product of Scheme 5 is treated with carbonyl di-imidazolein dichloromethane followed by an acid work-up to provide the carbonateester. Subsequent steps involve oxidation of the primary alcohol to thealdehyde and subsequently to the carboxylic acid followed bydeprotection to give a compound of formula I. Oxidation conditions toconvert the alcohol to the aldehyde include Parikh-Doering oxidation ofthe primary alcohol moiety using sulfur trioxide pyridine complex togive the corresponding aldehyde. Alternative oxidation agents to convertthe primary alcohol to the aldehyde include pyridinium dichromate (PDC),N-chlorosuccinimide (NCS)/benzenesulfenamide (PhSNHtBu) optionally inthe presence of 2-methyl-2-butene as a chlorine scavenger, RuCl₃/NaIO₄,tetramethylpiperidine N-oxide (TEMPO)/bisacetoxyiodobenzene(BIAB)/NaHCO₃, or 2-iodoxybenzoic acid (IBX). Oxidation conditions toconvert the aldehyde to the carboxylic include sodium or potassiumpermanganate. Sodium carbonate-mediated deprotection in methanolprovides the compound of formula I.

Alternatively, one-pot synthesis of the carboxylic acid can beaccomplished using tetrapropylammonium perruthenate (TPAP)/N-Methylmorpholine N-oxide (NMO) monohydrate as depicted in Scheme 8. Otheroxidants that can be used for this transformation includeOxone/TPAP/NMO/TBAB, and KMnO₄.

The protected carboxylic acid can be deprotected using a base to form acompound of formula I, as depicted in Scheme 9. Bases that can be usedfor this transformation include NaOH, Na₂CO₃, NaHCO₃, or Na₂CO₃/NaHCO₃.

Scheme 10 provides an alternative process for making compounds offormula I via a Sonagashira/cyclization protocol similar to thatdescribed in Scheme 3 and 4. From the silyl protected propargyl alcoholshown, conversion to the propargyl chloride followed by formation of theGrignard reagent and subsequent nucleophilic substitution provides((R₃-substituted-isopropyl ester, which is used in another step of thesynthesis. To complete the amine moiety, 4-nitro-3-R₂-aniline is firstbrominated, and then converted to the toluenesulfonic acid salt of(R)-1-(4-amino-2-bromo-5-R₂-substituted-phenylamino)-3-(benzyloxy)propan-2-olin a two-step process beginning with alkylation of the aniline aminogroup by (R)-2-(benzyloxymethyl)oxirane, followed by reduction of thenitro group to the corresponding amine. Palladium catalyzed coupling ofthe product with the R₃-substituted-isopropyl ester (discussed above)provides the intermediate akynyl compound, which is then cyclized to theindole moiety to produce the benzyl protected amine moiety. The sameprocess can be used from silyl-propargyl alcohol to give the benzylester. Subsequent coupling with1-(R-substituted-5-yl)cyclopropanecarbonyl chloride according to Scheme4 provides the isopropyl ester of a compound of Formula I.

The hydrolysis of the isopropyl ester of Scheme 11 provides the compoundof formula Ia. Bases that can be used for this transformation includealkali and alkali metal hydroxides; NaOH, or LiOH, for instance can beused.

Scheme 12 depicts the synthesis of a compound of formula Ia or IIa whereR₂ is F. In the first step, the diol is treated with carbonyldi-imidazole to protect the diol moiety as the carbonate ester and thenthe oxidant N-chlorosuccinimide (NCS)/benzenesulfenamide (PhSNHtBu),used optionally in the presence of 2-methyl-2-butene as a chlorinescavenger, provides the intermediate aldehyde. The intermediate aldehydeis converted to the compound of formula Ia wherein R₂ is F via treatmentwith permanganate, followed by deprotection in the presence of a basesuch as Na₂CO₃, to give the desired carboxylic acid as the sodium salt.Alternatively, the intermediate aldehyde is converted to the compound offormula II a, where R₂ is F, via treatment with a base such as Na₂CO₃,

Formulations, Administrations, and Uses

Accordingly, in another aspect of the present invention,pharmaceutically acceptable compositions are provided, wherein thesecompositions comprise any of the compounds as described herein, andoptionally comprise a pharmaceutically acceptable carrier, adjuvant orvehicle. In certain embodiments, these compositions optionally furthercomprise one or more additional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative or a prodrug thereof. Accordingto the present invention, a pharmaceutically acceptable derivative or aprodrug includes, but is not limited to, pharmaceutically acceptablesalts, esters, salts of such esters, or any other adduct or derivativewhich upon administration to a patient in need is capable of providing,directly or indirectly, a compound as otherwise described herein, or ametabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describes pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersible products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents; releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by ABC transporteractivity. In certain embodiments, the present invention provides amethod of treating a condition, disease, or disorder implicated by adeficiency of ABC transporter activity, the method comprisingadministering a composition comprising a compound of formulae (I or Ia)to a subject, preferably a mammal, in need thereof.

In certain preferred embodiments, the present invention provides amethod of treating Cystic fibrosis, emphysema, Hereditaryhemochromatosis, Coagulation-Fibrinolysis deficiencies, such as ProteinC deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism,Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma,Glycanosis CDG type 1, emphysema, Congenital hyperthyroidism,Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders asuch as Huntington,Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,Dentatorubal pallidoluysian, and Myotonic dystrophy, as well asSpongiform encephalopathies, such as Hereditary Creutzfeldt-Jakobdisease (due to Prion protein processing defect), Fabry disease,Straussler-Scheinker disease, secretory diarrhea, polycystic kidneydisease, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome, comprising the step of administering to saidmammal an effective amount of a composition comprising a compound offormulae (I or Ia), or a preferred embodiment thereof as set forthabove.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising a compound of formulae (I or Ia), or a preferred embodimentthereof as set forth above.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of Cystic fibrosis,emphysema, Hereditary hemochromatosis, Coagulation-Fibrinolysisdeficiencies, such as Protein C deficiency, Type 1 hereditaryangioedema, Lipid processing deficiencies, such as Familialhypercholesterolemia, Type 1 chylomicronemia, Abetalipoproteinemia,Lysosomal storage diseases, such as I-cell disease/Pseudo-Hurler,Mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism,Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma,Glycanosis CDG type 1, emphysema, Congenital hyperthyroidism,Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders asuch as Huntington,Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,Dentatorubal pallidoluysian, and Myotonic dystrophy, as well asSpongiform encephalopathies, such as Hereditary Creutzfeldt-Jakobdisease, Fabry disease, Straussler-Scheinker disease, secretorydiarrhea, polycystic kidney disease, chronic obstructive pulmonarydisease (COPD), dry eye disease, and Sjögren's Syndrome.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of Cystic fibrosis, emphysema, Hereditary hemochromatosis,Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency,Type 1 hereditary angioedema, Lipid processing deficiencies, such asFamilial hypercholesterolemia, Type 1 chylomicronemia,Abetalipoproteinemia, Lysosomal storage diseases, such as I-celldisease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetesmellitus, Laron dwarfism, Myleoperoxidase deficiency, Primaryhypoparathyroidism, Melanoma, Glycanosis CDG type 1, emphysema,Congenital hyperthyroidism, Osteogenesis imperfecta, Hereditaryhypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders asuch as Huntington, Spinocerebullar ataxia typeI, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, andMyotonic dystrophy, as well as Spongiform encephalopathies, such asHereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker disease, secretory diarrhea, polycystic kidneydisease, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

As described generally above, the compounds of the invention are usefulas modulators of ABC transporters. Thus, without wishing to be bound byany particular theory, the compounds and compositions are particularlyuseful for treating or lessening the severity of a disease, condition,or disorder where hyperactivity or inactivity of ABC transporters isimplicated in the disease, condition, or disorder. When hyperactivity orinactivity of an ABC transporter is implicated in a particular disease,condition, or disorder, the disease, condition, or disorder may also bereferred to as a “ABC transporter-mediated disease, condition ordisorder”. Accordingly, in another aspect, the present inventionprovides a method for treating or lessening the severity of a disease,condition, or disorder where hyperactivity or inactivity of an ABCtransporter is implicated in the disease state.

The activity of a compound utilized in this invention as a modulator ofan ABC transporter may be assayed according to methods describedgenerally in the art and in the Examples herein.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another agent used to treat the same disorder), orthey may achieve different effects (e.g., control of any adverseeffects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

In one embodiment, the additional therapeutic agent is selected from amucolytic agent, bronchodialator, an antibiotic, an anti-infectiveagent, an anti-inflammatory agent, a CFTR modulator other than acompound of formula I of the invention, or a nutritional agent.

In one embodiment, the additional therapeutic agent is an antibiotic.Exemplary antibiotics useful herein include tobramycin, includingtobramycin inhaled powder (TIP), azithromycin, aztreonam, including theaerosolized form of aztreonam, amikacin, including liposomalformulations thereof, ciprofloxacin, including formulations thereofsuitable for administration by inhalation, levoflaxacin, includingaerosolized formulations thereof, and combinations of two antibiotics,e.g., fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogenphosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent is a CFTR modulator otherthan a compound of formula I, i.e., an agent that has the effect ofmodulating CFTR activity. Exemplary such agents include ataluren(“PTC124®”; 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid),sinapultide, lancovutide, depelestat (a human recombinant neutrophilelastase inhibitor), and cobiprostone(7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid).

In another embodiment, the additional agent is a nutritional agent.Exemplary nutritional agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

In another embodiment, the additional agent is a compound selected fromgentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat,felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMPmodulators such as rolipram, sildenafil, milrinone, tadalafil, aminone,isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP 90inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin,lactacystin, etc.

In other embodiments, the additional agent is a compound disclosed in WO2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO2006101740. In another embodiment, the additional agent is abenzo[c]quinolizinium derivative that exhibits CFTR modulation activityor a benzopyran derivative that exhibits CFTR modulation activity. Inanother embodiment, the additional agent is a compound disclosed in U.S.Pat. No. 7,202,262, U.S. Pat. No. 6,992,096, US20060148864,US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456,WO2006044682, WO2006044505, WO2006044503, WO2006044502, or WO2004091502.In another embodiment, the additional agent is a compound disclosed inWO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006099256,WO2006127588, or WO2007044560. In another embodiment, the additionalagent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.

In one embodiment, 100 mg of a compound of formula I may be administeredto a subject in need thereof followed by co-administration of 150 mg ofN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide(Compound 2). In another embodiment, 100 mg of a compound of formula Imay be administered to a subject in need thereof followed byco-administration of 250 mg of Compound 2. In these embodiments, thedosage amounts may be achieved by administration of one or more tabletsof the invention. Compound 2 may be administered as a pharmaceuticalcomposition comprising Compound 2 and a pharmaceutically acceptablecarrier. The duration of administration may continue until ameliorationof the disease is achieved or until a subject's physician advises, e.g.duration of administration may be less than a week, 1 week, 2 weeks, 3weeks, or a month or longer. The co-administration period may bepreceded by an administration period of just a compound of formula Ialone. For example, there could be administration of 100 mg of Compound1 for 2 weeks followed by co-administration of 150 mg or 250 mg ofCompound 2 for 1 additional week.

In one embodiment, 100 mg of a compound of formula I may be administeredonce a day to a subject in need thereof followed by co-administration of150 mg of Compound 2 once a day. In another embodiment, 100 mg of acompound of formula I may be administered once a day to a subject inneed thereof followed by co-administration of 250 mg of Compound 2 oncea day. In these embodiments, the dosage amounts may be achieved byadministration of one or more tablets of the invention. Compound 2 maybe administered as a pharmaceutical composition comprising Compound 2and a pharmaceutically acceptable carrier. The duration ofadministration may continue until amelioration of the disease isachieved or until a subject's physician advises, e.g. duration ofadministration may be less than a week, 1 week, 2 weeks, 3 weeks, or amonth or longer. The co-administration period may be preceded by anadministration period of just a compound of formula I alone. Forexample, there could be administration of 100 mg of a compound offormula I for 2 weeks followed by co-administration of 150 mg or 250 mgof Compound 2 for 1 additional week.

In one embodiment, 100 mg of a compound of formula I may be administeredonce a day to a subject in need thereof followed by co-administration of150 mg of Compound 2 every 12 hours. In another embodiment, 100 mg of acompound of formula I may be administered once a day to a subject inneed thereof followed by co-administration of 250 mg of Compound 2 every12 hours. In these embodiments, the dosage amounts may be achieved byadministration of one or more tablets of the invention. Compound 2 maybe administered as a pharmaceutical composition comprising Compound 2and a pharmaceutically acceptable carrier. The duration ofadministration may continue until amelioration of the disease isachieved or until a subject's physician advises, e.g. duration ofadministration may be less than a week, 1 week, 2 weeks, 3 weeks, or amonth or longer. The co-administration period may be preceded by anadministration period of just a compound of formula I alone. Forexample, there could be administration of 100 mg of a compound offormula I for 2 weeks followed by co-administration of 150 mg or 250 mgof Compound 2 for 1 additional week.

These combinations are useful for treating the diseases described hereinincluding cystic fibrosis. These combinations are also useful in thekits described herein.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating ABC transporteractivity in a biological sample or a patient (e.g., in vitro or invivo), which method comprises administering to the patient, orcontacting said biological sample with a compound of formula I or acomposition comprising said compound. The term “biological sample”, asused herein, includes, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof.

Modulation of ABC transporter activity in a biological sample is usefulfor a variety of purposes that are known to one of skill in the art.Examples of such purposes include, but are not limited to, the study ofABC transporters in biological and pathological phenomena; and thecomparative evaluation of new modulators of ABC transporters.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with a compound of formulae I or Ia. Inpreferred embodiments, the anion channel is a chloride channel or abicarbonate channel. In other preferred embodiments, the anion channelis a chloride channel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional ABC transporters in amembrane of a cell, comprising the step of contacting said cell with acompound of formulae (I or Ia). The term “functional ABC transporter” asused herein means an ABC transporter that is capable of transportactivity. In preferred embodiments, said functional ABC transporter isCFTR.

According to another preferred embodiment, the activity of the ABCtransporter is measured by measuring the transmembrane voltagepotential. Means for measuring the voltage potential across a membranein the biological sample may employ any of the known methods in the art,such as optical membrane potential assay or other electrophysiologicalmethods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In another aspect the present invention provides a kit for use inmeasuring the activity of a ABC transporter or a fragment thereof in abiological sample in vitro or in vivo comprising (i) a compositioncomprising a compound of formulae (I or Ia) or any of the aboveembodiments; and (ii) instructions for a.) contacting the compositionwith the biological sample and b.) measuring activity of said ABCtransporter or a fragment thereof. In one embodiment, the kit furthercomprises instructions for a.) contacting an additional composition withthe biological sample; b.) measuring the activity of said ABCtransporter or a fragment thereof in the presence of said additionalcompound, and c.) comparing the activity of the ABC transporter in thepresence of the additional compound with the density of the ABCtransporter in the presence of a composition of formulae (I or Ia). Inpreferred embodiments, the kit is used to measure the density of CFTR.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES Reagents and Compounds

Vitride® (sodium bis(2-methoxyethoxy)aluminum hydride [orNaAlH₂(OCH₂CH₂OCH₃)₂], 65 wgt % solution in toluene) was purchased fromAldrich Chemicals. 3-Fluoro-4-nitroaniline was purchased from CapotChemicals. 5-Bromo-2,2-difluoro-1,3-benzodioxole was purchased from AlfaAesar. 2,2-Difluoro-1,3-benzodioxole-5-carboxylic acid was purchasedfrom Saltigo (an affiliate of the Lanxess Corporation).

Anywhere in the present application where a name of a compound may notcorrectly describe the structure of the compound, the structuresupersedes the name and governs.

Acid Chloride Moiety Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol

Commercially available 2,2-difluoro-1,3-benzodioxole-5-carboxylic acid(1.0 eq) is slurried in toluene (10 vol). Vitride® (2 eq) is added viaaddition funnel at a rate to maintain the temperature at 15-25° C. Atthe end of addition the temperature is increased to 40° C. for 2 h then10% (w/w) aq. NaOH (4.0 eq) is carefully added via addition funnelmaintaining the temperature at 40-50° C. After stirring for anadditional 30 minutes, the layers are allowed to separate at 40° C. Theorganic phase is cooled to 20° C. then washed with water (2×1.5 vol),dried (Na₂SO₄), filtered, and concentrated to afford crude(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol that is used directly inthe next step.

Synthesis of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole

(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol (1.0 eq) is dissolved inMTBE (5 vol). A catalytic amount of DMAP (1 mol %) is added and SOCl₂(1.2 eq) is added via addition funnel. The SOCl₂ is added at a rate tomaintain the temperature in the reactor at 15-25° C. The temperature isincreased to 30° C. for 1 hour then cooled to 20° C. then water (4 vol)is added via addition funnel maintaining the temperature at less than30° C. After stirring for an additional 30 minutes, the layers areallowed to separate. The organic layer is stirred and 10% (w/v) aq. NaOH(4.4 vol) is added. After stirring for 15 to 20 minutes, the layers areallowed to separate. The organic phase is then dried (Na₂SO₄), filtered,and concentrated to afford crude5-chloromethyl-2,2-difluoro-1,3-benzodioxole that is used directly inthe next step.

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

A solution of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole (1 eq) inDMSO (1.25 vol) is added to a slurry of NaCN (1.4 eq) in DMSO (3 vol)maintaining the temperature between 30-40° C. The mixture is stirred for1 hour then water (6 vol) is added followed by MTBE (4 vol). Afterstirring for 30 min, the layers are separated. The aqueous layer isextracted with MTBE (1.8 vol). The combined organic layers are washedwith water (1.8 vol), dried (Na₂SO₄), filtered, and concentrated toafford crude (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (95%) thatis used directly in the next step.

Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile

A stock solution of 50% w/w NaOH was degassed via nitrogen sparge for noless than 16 h. An appropriate amount of MTBE was similarly degassed forseveral hours. To a reactor purged with nitrogen was charged degassedMTBE (143 mL) followed by(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (40.95 g, 207.7 mmol)and tetrabutylammonium bromide (2.25 g, 10.38 mmol). The volume of themixture was noted and the mixture was degassed via nitrogen sparge for30 min. Enough degassed MTBE is charged to return the mixture to theoriginal volume prior to degassing. To the stirring mixture at 23.0° C.was charged degassed 50% w/w NaOH (143 mL) over 10 min followed by1-bromo-2-chloroethane (44.7 g, 311.6 mmol) over 30 min. The reactionwas analyzed by HPLC in 1 h intervals for % conversion. Before sampling,stirring was stopped and the phases allowed to separate. The top organicphase was sampled for analysis. When a % conversion >99% was observed(typically after 2.5-3 h), the reaction mixture was cooled to 10° C. andwas charged with water (461 mL) at such a rate as to maintain atemperature <25° C. The temperature was adjusted to 20-25° C. and thephases separated. Note: sufficient time should be allowed for completephase separation. The aqueous phase was extracted with MTBE (123 mL),and the combined organic phase was washed with 1 N HCl (163 mL) and 5%NaCl (163 mL). The solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile in MTBE wasconcentrated to 164 mL under vacuum at 40-50° C. The solution wascharged with ethanol (256 mL) and again concentrated to 164 mL undervacuum at 50-60° C. Ethanol (256 mL) was charged and the mixtureconcentrated to 164 mL under vacuum at 50-60° C. The resulting mixturewas cooled to 20-25° C. and diluted with ethanol to 266 mL inpreparation for the next step. ¹H NMR (500 MHz, DMSO) δ 7.43 (d, J=8.4Hz, 1H), 7.40 (d, J=1.9 Hz, 1H), 7.30 (dd, J=8.4, 1.9 Hz, 1H), 1.75 (m,2H), 1.53 (m, 2H).

Synthesis of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid

The solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile in ethanolfrom the previous step was charged with 6 N NaOH (277 mL) over 20 minand heated to an internal temperature of 77-78° C. over 45 min. Thereaction progress was monitored by HPLC after 16 h. Note: theconsumption of both(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile and theprimary amide resulting from partial hydrolysis of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile weremonitored. When a % conversion >99% was observed (typically 100%conversion after 16 h), the reaction mixture was cooled to 25° C. andcharged with ethanol (41 mL) and DCM (164 mL) The solution was cooled to10° C. and charged with 6 N HCl (290 mL) at such a rate as to maintain atemperature <25° C. After warming to 20-25° C., the phases were allowedto separate. The bottom organic phase was collected and the top aqueousphase was back extracted with DCM (164 mL) Note: the aqueous phase wassomewhat cloudy before and after the extraction due to a highconcentration of inorganic salts. The organics were combined andconcentrated under vacuum to 164 mL. Toluene (328 mL) was charged andthe mixture condensed to 164 mL at 70-75° C. The mixture was cooled to45° C., charged with MTBE (364 mL) and stirred at 60° C. for 20 min. Thesolution was cooled to 25° C. and polish filtered to remove residualinorganic salts. MTBE (123 mL) was used to rinse the reactor and thecollected solids. The combined organics were transferred to a cleanreactor in preparation for the next step.

Isolation of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid

The solution of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid fromthe previous step is concentrated under vacuum to 164 mL, charged withtoluene (328 mL) and concentrated to 164 mL at 70-75° C. The mixture wasthen heated to 100-105° C. to give a homogeneous solution. Afterstirring at that temperature for 30 min, the solution was cooled to 5°C. over 2 hours and maintained at 5° C. for 3 hours. The mixture wasthen filtered and the reactor and collected solid washed with cold 1:1toluene/n-heptane (2×123 mL). The material was dried under vacuum at 55°C. for 17 hours to provide1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid as anoff-white crystalline solid.1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid wasisolated in 79% yield from(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (3 steps includingisolation) and with an HPLC purity of 99.0% AUC. ESI-MS m/z calc.242.04. found 241.58 (M+1)⁺; ¹H NMR (500 MHz, DMSO) δ 12.40 (s, 1H),7.40 (d, J=1.6 Hz, 1H), 7.30 (d, J=8.3 Hz, 1H), 7.17 (dd, J=8.3, 1.7 Hz,1H), 1.46 (m, 2H), 1.17 (m, 2H).

Alternative Synthesis of the Acid Chloride Moiety Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile

A reactor was purged with nitrogen and charged with 900 mL of toluene.The solvent was degassed via nitrogen sparge for no less than 16 h. Tothe reactor was then charged Na₃PO₄ (155.7 g, 949.5 mmol), followed bybis(dibenzylideneacetone) palladium (0) (7.28 g, 12.66 mmol). A 10% w/wsolution of tert-butylphosphine in hexanes (51.23 g, 25.32 mmol) wascharged over 10 min at 23° C. from a nitrogen purged addition funnel.The mixture was allowed to stir for 50 min, at which time5-bromo-2,2-difluoro-1,3-benzodioxole (75 g, 316.5 mmol) was added over1 min. After stirring for an additional 50 min, the mixture was chargedwith ethyl cyanoacetate (71.6 g, 633.0 mmol) over 5 min followed bywater (4.5 mL) in one portion. The mixture was heated to 70° C. over 40min and analyzed by HPLC every 1-2 h for the percent conversion of thereactant to the product. After conversion was observed (typically 100%conversion after 5-8 h), the mixture was cooled to 20-25° C. andfiltered through a celite pad. The celite pad was rinsed with toluene(2×450 mL) and the combined organics were concentrated to 300 mL undervacuum at 60-65° C. The concentrate was charged with 225 mL DMSO andconcentrated under vacuum at 70-80° C. until active distillation of thesolvent ceased. The solution was cooled to 20-25° C. and diluted to 900mL with DMSO in preparation for Step 2. ¹H NMR (500 MHz, CDCl₃) δ7.16-7.10 (m, 2H), 7.03 (d, J=8.2 Hz, 1H), 4.63 (s, 1H), 4.19 (m, 2H),1.23 (t, J=7.1 Hz, 3H).

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

The DMSO solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile fromabove was charged with 3 N HCl (617.3 mL, 1.85 mol) over 20 min whilemaintaining an internal temperature <40° C. The mixture was then heatedto 75° C. over 1 h and analyzed by HPLC every 1-2 h for % conversion.When a conversion of >99% was observed (typically after 5-6 h), thereaction was cooled to 20-25° C. and extracted with MTBE (2×525 mL),with sufficient time to allow for complete phase separation during theextractions. The combined organic extracts were washed with 5% NaCl(2×375 mL). The solution was then transferred to equipment appropriatefor a 1.5-2.5 Torr vacuum distillation that was equipped with a cooledreceiver flask. The solution was concentrated under vacuum at <60° C. toremove the solvents. (2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrilewas then distilled from the resulting oil at 125-130° C. (oventemperature) and 1.5-2.0 Torr.(2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrile was isolated as a clearoil in 66% yield from 5-bromo-2,2-difluoro-1,3-benzodioxole (2 steps)and with an HPLC purity of 91.5% AUC (corresponds to a w/w assay of95%). ¹H NMR (500 MHz, DMSO) δ 7.44 (br s, 1H), 7.43 (d, J=8.4 Hz, 1H),7.22 (dd, J=8.2, 1.8 Hz, 1H), 4.07 (s, 2H).

The remaining steps are the same as described above for the synthesis ofthe acid moiety.

Amine Moiety Synthesis of 2-bromo-5-fluoro-4-nitroaniline

A flask was charged with 3-fluoro-4-nitroaniline (1.0 equiv) followed byethyl acetate (10 vol) and stirred to dissolve all solids.N-Bromosuccinimide (1.0 equiv) was added as a portion-wise as tomaintain internal temperature of 22° C. At the end of the reaction, thereaction mixture was concentrated in vacuo on a rotavap. The residue wasslurried in distilled water (5 vol) to dissolve and remove succinimide.(The succinimide can also be removed by water workup procedure.) Thewater was decanted and the solid was slurried in 2-propanol (5 vol)overnight. The resulting slurry was filtered and the wetcake was washedwith 2-propanol, dried in vacuum oven at 50° C. overnight with N₂ bleeduntil constant weight was achieved. A yellowish tan solid was isolated(50% yield, 97.5% AUC). Other impurities were a bromo-regioisomer (1.4%AUC) and a di-bromo adduct (1.1% AUC). ¹H NMR (500 MHz, DMSO) δ 8.19(1H, d, J=8.1 Hz), 7.06 (br. s, 2H), 6.64 (d, 1H, J=14.3 Hz).

Synthesis of benzylglycolated-4-ammonium-2-bromo-5-fluoroanilinetosylate salt

A thoroughly dried flask under N₂ was charged with the following:Activated powdered 4A molecular sieves (50 wt % based on2-bromo-5-fluoro-4-nitroaniline), 2-Bromo-5-fluoro-4-nitroaniline (1.0equiv), zinc perchlorate dihydrate (20 mol %), and toluene (8 vol). Themixture was stirred at room temperature for NMT 30 min. Lastly,(R)-benzyl glycidyl ether (2.0 equiv) in toluene (2 vol) was added in asteady stream. The reaction was heated to 80° C. (internal temperature)and stirred for approximately 7 hours or until2-Bromo-5-fluoro-4-nitroaniline was <5% AUC.

The reaction was cooled to room temperature and Celite (50 wt %) wasadded, followed by ethyl acetate (10 vol). The resulting mixture wasfiltered to remove Celite and sieves and washed with ethyl acetate (2vol). The filtrate was washed with ammonium chloride solution (4 vol,20% w/v). The organic layer was washed with sodium bicarbonate solution(4 vol×2.5% w/v). The organic layer was concentrated in vacuo on arotovap. The resulting slurry was dissolved in isopropyl acetate (10vol) and this solution was transferred to a Buchi hydrogenator.

The hydrogenator was charged with 5 wt % Pt(S)/C (1.5 mol %) and themixture was stirred under N₂ at 30° C. (internal temperature). Thereaction was flushed with N₂ followed by hydrogen. The hydrogenatorpressure was adjusted to 1 Bar of hydrogen and the mixture was stirredrapidly (>1200 rpm). At the end of the reaction, the catalyst wasfiltered through a pad of Celite and washed with dichloromethane (10vol). The filtrate was concentrated in vacuo. Any remaining isopropylacetate was chased with dichloromethane (2 vol) and concentrated on arotavap to dryness.

The resulting residue was dissolved in dichloromethane (10 vol).p-Toluenesulfonic acid monohydrate (1.2 equiv) was added and stirredovernight. The product was filtered and washed with dichloromethane (2vol) and suction dried. The wetcake was transferred to drying trays andinto a vacuum oven and dried at 45° C. with N₂ bleed until constantweight was achieved. Benzylglycolated-4-ammonium-2-bromo-5-fluoroanilinetosylate salt was isolated as an off-white solid.

Chiral purity was determined to be >97% ee.

Synthesis of (3-Chloro-3-methylbut-1-ynyl)trimethylsilane

Propargyl alcohol (1.0 equiv) was charged to a vessel. Aqueoushydrochloric acid (37%, 3.75 vol) was added and stirring begun. Duringdissolution of the solid alcohol, a modest endotherm (5-6° C.) isobserved. The resulting mixture was stirred overnight (16 h), slowlybecoming dark red. A 30 L jacketed vessel is charged with water (5 vol)which is then cooled to 10° C. The reaction mixture is transferredslowly into the water by vacuum, maintaining the internal temperature ofthe mixture below 25° C. Hexanes (3 vol) is added and the resultingmixture is stirred for 0.5 h. The phases were settled and the aqueousphase (pH<1) was drained off and discarded. The organic phase wasconcentrated in vacuo using a rotary evaporator, furnishing the productas red oil.

Synthesis of (4-(Benzyloxy)-3,3-dimethylbut-1-ynyl)trimethylsilane

Method A

All equivalent and volume descriptors in this part are based on a 250 greaction. Magnesium turnings (69.5 g, 2.86 mol, 2.0 equiv) were chargedto a 3 L 4-neck reactor and stirred with a magnetic stirrer undernitrogen for 0.5 h. The reactor was immersed in an ice-water bath. Asolution of the propargyl chloride (250 g, 1.43 mol, 1.0 equiv) in THF(1.8 L, 7.2 vol) was added slowly to the reactor, with stirring, untilan initial exotherm (˜10° C.) was observed. The Grignard reagentformation was confirmed by IPC using ¹H-NMR spectroscopy. Once theexotherm subsided, the remainder of the solution was added slowly,maintaining the batch temperature <15° C. The addition required ˜3.5 h.The resulting dark green mixture was decanted into a 2 L capped bottle.

All equivalent and volume descriptors in this part are based on a 500 greaction. A 22 L reactor was charged with a solution of benzylchloromethyl ether (95%, 375 g, 2.31 mol, 0.8 equiv) in THF (1.5 L, 3vol). The reactor was cooled in an ice-water bath. Two Grignard reagentbatches prepared as described above were combined and then added slowlyto the benzyl chloromethyl ether solution via an addition funnel,maintaining the batch temperature below 25° C. The addition required 1.5h. The reaction mixture was stirred overnight (16 h).

All equivalent and volume descriptors in this part are based on a 1 kgreaction. A solution of 15% ammonium chloride was prepared in a 30 Ljacketed reactor (1.5 kg in 8.5 kg of water, 10 vol). The solution wascooled to 5° C. Two Grignard reaction mixtures prepared as describedabove were combined and then transferred into the ammonium chloridesolution via a header vessel. An exotherm was observed in this quench,which was carried out at a rate such as to keep the internal temperaturebelow 25° C. Once the transfer was complete, the vessel jackettemperature was set to 25° C. Hexanes (8 L, 8 vol) was added and themixture was stirred for 0.5 h. After settling the phases, the aqueousphase (pH 9) was drained off and discarded. The remaining organic phasewas washed with water (2 L, 2 vol). The organic phase was concentratedin vacuo using a 22 L rotary evaporator, providing the crude product asan orange oil.

Method B

Magnesium turnings (106 g, 4.35 mol, 1.0 eq) were charged to a 22 Lreactor and then suspended in THF (760 mL, 1 vol). The vessel was cooledin an ice-water bath such that the batch temperature reached 2° C. Asolution of the propargyl chloride (760 g, 4.35 mol, 1.0 equiv) in THF(4.5 L, 6 vol) was added slowly to the reactor. After 100 mL was added,the addition was stopped and the mixture stirred until a 13° C. exothermwas observed, indicating the Grignard reagent initiation. Once theexotherm subsided, another 500 mL of the propargyl chloride solution wasadded slowly, maintaining the batch temperature <20° C. The Grignardreagent formation was confirmed by IPC using ¹H-NMR spectroscopy. Theremainder of the propargyl chloride solution was added slowly,maintaining the batch temperature <20° C. The addition required ˜1.5 h.The resulting dark green solution was stirred for 0.5 h. The Grignardreagent formation was confirmed by IPC using ¹H-NMR spectroscopy. Neatbenzyl chloromethyl ether was charged to the reactor addition funnel andthen added dropwise into the reactor, maintaining the batch temperaturebelow 25° C. The addition required 1.0 h. The reaction mixture wasstirred overnight. The aqueous work-up and concentration was carried outusing the same procedure and relative amounts of materials as in MethodA to give the product as an orange oil.

Synthesis of 4-Benzyloxy-3,3-dimethylbut-1-yne

A 30 L jacketed reactor was charged with methanol (6 vol) which was thencooled to 5° C. Potassium hydroxide (85%, 1.3 equiv) was added to thereactor. A 15-20° C. exotherm was observed as the potassium hydroxidedissolved. The jacket temperature was set to 25° C. A solution of4-benzyloxy-3,3-dimethyl-1-trimethylsilylbut-1-yne (1.0 equiv) inmethanol (2 vol) was added and the resulting mixture was stirred untilreaction completion, as monitored by HPLC. Typical reaction time at 25°C. is 3-4 h. The reaction mixture is diluted with water (8 vol) and thenstirred for 0.5 h. Hexanes (6 vol) was added and the resulting mixturewas stirred for 0.5 h. The phases were allowed to settle and then theaqueous phase (pH 10-11) was drained off and discarded. The organicphase was washed with a solution of KOH (85%, 0.4 equiv) in water (8vol) followed by water (8 vol). The organic phase was then concentrateddown using a rotary evaporator, yielding the title material as ayellow-orange oil. Typical purity of this material is in the 80% rangewith primarily a single impurity present. ¹H NMR (400 MHz, C₆D₆) δ 7.28(d, 2H, J=7.4 Hz), 7.18 (t, 2H, J=7.2 Hz), 7.10 (d, 1H, J=7.2 Hz), 4.35(s, 2H), 3.24 (s, 2H), 1.91 (s, 1H), 1.25 (s, 6H).

Synthesis ofN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindoleMethod A Synthesis of Benzylglycolated4-Amino-2-(4-benzyloxy-3,3-dimethylbut-1-ynyl)-5-fluoroaniline

Benzylglycolated 4-ammonium-2-bromo-5-fluoroaniline tosylate salt wasfreebased by stirring the solid in EtOAc (5 vol) and saturated NaHCO₃solution (5 vol) until clear organic layer was achieved. The resultinglayers were separated and the organic layer was washed with saturatedNaHCO₃ solution (5 vol) followed by brine and concentrated in vacuo toobtain benzylglocolated 4-ammonium-2-bromo-5-fluoroaniline tosylate saltas an oil.

Then, a flask was charged with benzylglycolated4-ammonium-2-bromo-5-fluoroaniline tosylate salt (freebase, 1.0 equiv),Pd(OAc) (4.0 mol %), dppb (6.0 mol %) and powdered K₂CO₃ (3.0 equiv) andstirred with acetonitrile (6 vol) at room temperature. The resultingreaction mixture was degassed for approximately 30 min by bubbling in N₂with vent. Then 4-benzyloxy-3,3-dimethylbut-1-yne (1.1 equiv) dissolvedin acetonitrile (2 vol) was added in a fast stream and heated to 80° C.and stirred until complete consumption of4-ammonium-2-bromo-5-fluoroaniline tosylate salt was achieved. Thereaction slurry was cooled to room temperature and filtered through apad of Celite and washed with acetonitrile (2 vol). Filtrate wasconcentrated in vacuo and the residue was redissolved in EtOAc (6 vol).The organic layer was washed twice with NH₄Cl solution (20% w/v, 4 vol)and brine (6 vol). The resulting organic layer was concentrated to yieldbrown oil and used as is in the next reaction.

Synthesis ofN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindole

Crude oil of benzylglycolated4-amino-2-(4-benzyloxy-3,3-dimethylbut-1-ynyl)-5-fluoroaniline wasdissolved in acetonitrile (6 vol) and added (MeCN)₂PdCl₂ (15 mol %) atroom temperature. The resulting mixture was degassed using N₂ with ventfor approximately 30 min. Then the reaction mixture was stirred at 80°C. under N₂ blanket overnight. The reaction mixture was cooled to roomtemperature and filtered through a pad of Celite and washed the cakewith acetonitrile (1 vol). The resulting filtrate was concentrated invacuo and redissolved in EtOAc (5 vol). Deloxane-II THP (5 wt % based onthe theoretical yield ofN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindole)was added and stirred at room temperature overnight. The mixture wasthen filtered through a pad of silica (2.5 inch depth, 6 inch diameterfilter) and washed with EtOAc (4 vol). The filtrate was concentrateddown to a dark brown residue, and used as is in the next reaction.

Repurification of crudeN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindole:

The crudeN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindolewas dissolved in dichloromethane (˜1.5 vol) and filtered through a padof silica initially using 30% EtOAc/heptane where impurities werediscarded. Then the silica pad was washed with 50% EtOAc/heptane toisolateN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindoleuntil faint color was observed in the filtrate. This filtrate wasconcentrated in vacuo to afford brown oil which crystallized on standingat room temperature. ¹H NMR (400 MHz, DMSO) δ 7.38-7.34 (m, 4H),7.32-7.23 (m, 6H), 7.21 (d, 1H, J=12.8 Hz), 6.77 (d, 1H, J=9.0 Hz), 6.06(s, 1H), 5.13 (d, 1H, J=4.9 Hz), 4.54 (s, 2H), 4.46 (br. s, 2-H), 4.45(s, 2H), 4.33 (d, 1H, J=12.4 Hz), 4.09-4.04 (m, 2H), 3.63 (d, 1H, J=9.2Hz), 3.56 (d, 1H, J=9.2 Hz), 3.49 (dd, 1H, J=9.8, 4.4 Hz), 3.43 (dd, 1H,J=9.8, 5.7 Hz), 1.40 (s, 6H).

Synthesis ofN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindoleMethod B

Palladium acetate (33 g, 0.04 eq), dppb (94 g, 0.06 eq), and potassiumcarbonate (1.5 kg, 3.0 eq) are charged to a reactor. The free based oilbenzylglocolated 4-ammonium-2-bromo-5-fluoroaniline (1.5 kg, 1.0 eq) wasdissolved in acetonitrile (8.2 L, 4.1 vol) and then added to thereactor. The mixture was sparged with nitrogen gas for NLT 1 h. Asolution of 4-benzyloxy-3,3-dimethylbut-1-yne (70%, 1.1 kg, 1.05 eq) inacetonitrile was added to the mixture which was then sparged withnitrogen gas for NLT 1 h. The mixture was heated to 80° C. and thenstirred overnight. IPC by HPLC is carried out and the reaction isdetermined to be complete after 16 h. The mixture was cooled to ambienttemperature and then filtered through a pad of Celite (228 g). Thereactor and Celite pad were washed with acetonitrile (2×2 L, 2 vol). Thecombined phases are concentrated on a 22 L rotary evaporator until 8 Lof solvent have been collected, leaving the crude product in 7 L (3.5vol) of acetonitrile.

Bis-acetonitriledichloropalladium (144 g, 0.15 eq) was charged to thereactor. The crude solution was transferred back into the reactor andthe roto-vap bulb was washed with acetonitrile (4 L, 2 vol). Thecombined solutions were sparged with nitrogen gas for NLT 1 h. Thereaction mixture was heated to 80° C. for NLT 16 h. In process controlby HPLC shows complete consumption of starting material. The reactionmixture was filtered through Celite (300 g). The reactor and filter cakewere washed with acetonitrile (3 L, 1.5 vol). The combined filtrateswere concentrated to an oil by rotary evaporation. The oil was dissolvedin ethyl acetate (8.8 L, 4.4 vol). The solution was washed with 20%ammonium chloride (5 L, 2.5 vol) followed by 5% brine (5 L, 2.5 vol).Silica gel (3.5 kg, 1.8 wt. eq.) of silica gel was added to the organicphase, which was stirred overnight. Deloxan THP II metal scavenger (358g) and heptane (17.6 L) were added and the resulting mixture was stirredfor NLT 3 h. The mixture was filtered through a sintered glass funnel.The filter cake was washed with 30% ethyl acetate in heptane (25 L). Thecombined filtrates were concentrated under reduced pressure to giveN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindoleas a brown paste (1.4 kg).

Synthesis of benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide

1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid (1.3equiv) was slurried in toluene (2.5 vol, based on1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid) andthe mixture was heated to 60° C. SOCl₂ (1.7 equiv) was added viaaddition funnel. The resulting mixture was stirred for 2 hr. The tolueneand the excess SOCl₂ were distilled off using rotavop. Additionaltoluene (2.5 vol, based on1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid) wasadded and distilled again. The crude acid chloride was dissolved indichloromethane (2 vol) and added via addition funnel to a mixture ofN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindole(1.0 equiv), and triethylamine (2.0 equiv) in dichloromethane (7 vol)while maintaining 0-3° C. (internal temperature). The resulting mixturewas stirred at 0° C. for 4 hrs and then warmed to room temperatureovernight. Distilled water (5 vol) was added to the reaction mixture andstirred for NLT 30 min and the layers were separated. The organic phasewas washed with 20 wt % K₂CO₃ (4 vol×2) followed by a brine wash (4 vol)and concentrated to afford crude benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-ypcyclopropanecarboxamideas a thick brown oil, which was purified further using silica padfiltration.

Silica Gel Pad Filtration:

Crude benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamidewas dissolved in ethyl acetate (3 vol) in the presence of activatedcarbon Darco-G (10 wt %, based on theoretical yield of benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide)and stirred at room temperature overnight. To this mixture was addedheptane (3 vol) and filtered through a pad of silica gel (2×weight ofcrude benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide).The silica pad was washed with ethyl acetate/heptane (1:1, 6 vol) oruntil little color was detected in the filtrate. The filtrate wasconcentrated in vacuo to afford benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideas viscous reddish brown oil, and used directly in the next step.

Repurification:

Benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamidewas redissolved in dichloromethane (1 vol, based on theoretical yield ofbenzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide)and loaded onto a silica gel pad (2×weight of crude benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide).The silica pad was washed with dichloromethane (2 vol, based ontheoretical yield of benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide)and the filtrate was discarded. The silica pad was washed with 30% ethylacetate/heptane (5 vol) and the filtrate was concentrated in vacuo toafford benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideas viscous reddish orange oil, and used directly in the next step.

Synthesis of(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide

Method A

A 20 L autoclave was flushed three times with nitrogen gas and thencharged with palladium on carbon (Evonik E 101 NN/W, 5% Pd, 60% wet, 200g, 0.075 mol, 0.04 equiv). The autoclave was then flushed with nitrogenthree times. A solution of crude benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(1.3 kg, ˜1.9 mol) in THF (8 L, 6 vol) was added to the autoclave viasuction. The vessel was capped and then flushed three times withnitrogen gas. With gentle stirring, the vessel was flushed three timeswith hydrogen gas, evacuating to atmosphere by diluting with nitrogen.The autoclave was pressurized to 3 Bar with hydrogen and the agitationrate was increased to 800 rpm. Rapid hydrogen uptake was observed(dissolution). Once uptake subsided, the vessel was heated to 50° C.

For safety purposes, the thermostat was shut off at the end of everywork-day. The vessel was pressurized to 4 Bar with hydrogen and thenisolated from the hydrogen tank.

After 2 full days of reaction, more Pd/C (60 g, 0.023 mol, 0.01 equiv)was added to the mixture. This was done by flushing three times withnitrogen gas and then adding the catalyst through the solids additionport. Resuming the reaction was done as before. After 4 full days, thereaction was deemed complete by HPLC by the disappearance of not onlythe starting material but also of the peak corresponding to amono-benzylated intermediate.

The reaction mixture was filtered through a Celite pad. The vessel andfilter cake were washed with THF (2 L, 1.5 vol). The Celite pad was thenwetted with water and the cake discarded appropriately. The combinedfiltrate and THF wash were concentrated using a rotary evaporatoryielding the crude product as a black oil, 1 kg.

The equivalents and volumes in the following purification are based on 1kg of crude material. The crude black oil was dissolved in 1:1 ethylacetate-heptane. The mixture was charged to a pad of silica gel (1.5 kg,1.5 wt. equiv) in a fitted funnel that had been saturated with 1:1 ethylacetate-heptane. The silica pad was flushed first with 1:1 ethylacetate-heptane (6 L, 6 vol) and then with pure ethyl acetate (14 L, 14vol). The eluent was collected in 4 fractions which were analyzed byHPLC.

The equivalents and volumes in the following purification are based on0.6 kg of crude material. Fraction 3 was concentrated by rotaryevaporation to give a brown foam (600 g) and then redissolved in MTBE(1.8 L, 3 vol). The dark brown solution was stirred overnight at ambienttemperature, during which time, crystallization occurred. Heptane (55mL, 0.1 vol) was added and the mixture was stirred overnight. Themixture was filtered using a Buchner funnel and the filter cake waswashed with 3:1 MTBE-heptane (900 mL, 1.5 vol). The filter cake wasair-dried for 1 h and then vacuum dried at ambient temperature for 16 h,furnishing 253 g of (R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideas an off-white solid.

The equivalents and volumes for the following purification are based on1.4 kg of crude material. Fractions 2 and 3 from the above silica gelfiltration as well as material from a previous reaction were combinedand concentrated to give 1.4 kg of a black oil. The mixture wasresubmitted to the silica gel filtration (1.5 kg of silica gel, elutedwith 3.5 L, 2.3 vol of 1:1 ethyl acetate-heptane then 9 L, 6 vol of pureethyl acetate) described above, which upon concentration gave a tanfoamy solid (390 g).

The equivalents and volumes for the following purification are based on390 g of crude material. The tan solid was insoluble in MTBE, so wasdissolved in methanol (1.2 L, 3 vol). Using a 4 L Morton reactorequipped with a long-path distillation head, the mixture was distilleddown to 2 vol. MTBE (1.2 L, 3 vol) was added and the mixture wasdistilled back down to 2 vol. A second portion of MTBE (1.6 L, 4 vol)was added and the mixture was distilled back down to 2 vol. A thirdportion of MTBE (1.2 L, 3 vol) was added and the mixture was distilledback down to 3 vol. Analysis of the distillate by GC revealed it toconsist of ˜6% methanol. The thermostat was set to 48° C. (below theboiling temp of the MTBE-methanol azeotrope, which is 52° C.). Themixture was cooled to 20° C. over 2 h, during which time a relativelyfast crystallization occurred. After stirring the mixture for 2 h,heptane (20 mL, 0.05 vol) was added and the mixture was stirredovernight (16 h). The mixture was filtered using a Buchner funnel andthe filter cake was washed with 3:1 MTBE-heptane (800 mL, 2 vol). Thefilter cake was air-dried for 1 hand then vacuum dried at ambienttemperature for 16 h, furnishing 130 g of(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideas an off-white solid.

Method B

Benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamidewas dissolved in THF (3 vol) and then stripped to dryness to remove anyresidual solvent. Benzyl protected(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamidewas redissolved in THF (4 vol) and added to the hydrogenator containing5 wt % Pd/C (2.5 mol %, 60% wet, Degussa E5 E101 NN/W). The internaltemperature of the reaction was adjusted to 50° C., and flushed with N₂(x5) followed by hydrogen (x3). The hydrogenator pressure was adjustedto 3 Bar of hydrogen and the mixture was stirred rapidly (>1100 rpm). Atthe end of the reaction, the catalyst was filtered through a pad ofCelite and washed with THF (1 vol). The filtrate was concentrated invacuo to obtain a brown foamy residue. The resulting residue wasdissolved in MTBE (5 vol) and 0.5N HCl solution (2 vol) and distilledwater (1 vol) were added. The mixture was stirred for NLT 30 min and theresulting layers were separated. The organic phase was washed with 10 wt% K₂CO₃ solution (2 vol×2) followed by a brine wash. The organic layerwas added to a flask containing silica gel (25 wt %), Deloxan-THP II (5wt %, 75% wet), and Na₂SO₄ and stirred overnight. The resulting mixturewas filtered through a pad of Celite and washed with 10% THF/MTBE (3vol). The filtrate was concentrated in vacuo to afford crude(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideas pale tan foam.

(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamiderecovery from the mother liquor: Option A

Silica Gel Pad Filtration:

The mother liquor was concentrated in vacuo to obtain a brown foam,dissolved in dichloromethane (2 vol), and filtered through a pad ofsilica (3×weight of the crude(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide).The silica pad was washed with ethyl acetate/heptane (1:1, 13 vol) andthe filtrate was discarded. The silica pad was washed with 10% THF/ethylacetate (10 vol) and the filtrate was concentrated in vacuo to afford(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideas pale tan foam. The above crystallization procedure was followed toisolate the remaining(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.

(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamiderecovery from the mother liquor: Option B

Silica Gel Column Chromatography:

After chromatography on silica gel (50% ethyl acetate/hexanes to 100%ethyl acetate), the desired compound was isolated as pale tan foam. Theabove crystallization procedure was followed to isolate the remaining(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.

Additional Recrystallization of(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide

Solid(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(1.35 kg) was suspended in IPA (5.4 L, 4 vol) and then heated to 82° C.Upon complete dissolution (visual), heptane (540 mL, 0.4 vol) was addedslowly. The mixture was cooled to 58° C. The mixture was then cooledslowly to 51° C., during which time crystallization occurs. The heatsource was shut down and the recrystallization mixture was allowed tocool naturally overnight. The mixture was filtered using a benchtopBuchner funnel and the filter cake was washed with IPA (2.7 L, 2 vol).The filter cake was dried in the funnel under air flow for 8 h and thenwas oven-dried in vacuo at 45-50° C. overnight to give 1.02 kg ofrecrystallized(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.LC/MS (M+1) 521.5. LC/RT (min) 1.69. ¹H NMR (400.0 MHz, CD₃CN) d 7.69(d, J=7.7 Hz, 1H), 7.44 (d, J=1.6 Hz, 1H), 7.39 (dd, J=1.7, 8.3 Hz, 1H),7.31 (s, 1H), 7.27 (d, J=8.3 Hz, 1H), 7.20 (d, J=12.0 Hz, 1H), 6.34 (s,1H), 4.32 (d, J=6.8 Hz, 2H), 4.15-4.09 (m, 1H), 3.89 (dd, J=6.0, 11.5Hz, 1H), 3.63-3.52 (m, 3H), 3.42 (d, J=4.6 Hz, 1H), 3.21 (dd, J=6.2, 7.2Hz, 1H), 3.04 (t, J=5.8 Hz, 1H), 1.59 (dd, J=3.8, 6.8 Hz, 2H), 1.44 (s,3H), 1.33 (s, 3H) and 1.18 (dd, J=3.7, 6.8 Hz, 2H) ppm.

(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamidemay also be prepared by one of several synthetic routes disclosed in USpublished patent application US20090131492, incorporated herein byreference.

Synthesis of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-((4R)-8-fluoro-2-hydroxy-4-(hydroxymethyl)-1,1-dimethyl-1,2,4,5-tetrahydro-[1,4]oxazepino[4,5-a]indol-9-yl)cyclopropanecarboxamide

Method A

(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(11.5 mmol, 1 equiv) was suspended in DCM (51 mL, 8.5 vol). A solutionof Dess-Martin periodinane (0.3 M in DCM, 12.8 mmol, 1.1 equiv) wasadded at ambient temperature. The mixture was stirred until the reactionwas deemed complete by HPLC. A 5% aqueous solution of sodium sulfite wasadded and the mixture was stirred for up to 4 h. The phases wereseparated and then the organic phase was washed with 1 N HCl, brine andwas then concentrated by rotary evaporation. The residue was purified bychromatography. The yield of purified material was between 7 and 15%.

Method B

(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(48.03 mmol, 1 equiv) was dissolved in ethyl acetate (1.25 L, 50 vol)and heated. Silica-supported pyridinium dichromate (Si-PDC, 48.03 mmol,1 equiv) was charged to the stirring hot solution. The reaction wasstirred until deemed complete by HPLC. The reaction mixture was filteredthrough a pad of silica gel and the filter cake washed with ethylacetate (2×100 mL, 2×4 vol). The mother liquor was concentrated byrotary evaporation and the residue was purified by chromatography. Theyield of the purified material was 13.5%.

Synthesis of(R)-2-(5-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-1-(2,3-dihydroxypropyl)-6-fluoro-1H-indol-2-yl)-2-methylpropanoicacid

3.62 g of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-((4R)-8-fluoro-2-hydroxy-4-(hydroxymethyl)-1,1-dimethyl-1,2,4,5-tetrahydro-[1,4]oxazepino[4,5-α]indol-9-yl)cyclopropanecarboxamidewas charged in a 1 L flask along with 600 mL of toluene and stirred todissolution. 14.5 g of Ag₂CO₃ in Celite. The heterogenous suspension washeated to 90° C. and held for 7 hours at this temperature. Thesuspension was then allowed to cool naturally to ambient temperaturesand filtered over celite. The celite was washed with ethyl acetate untilno product comes off by HPLC, giving 1.24 g of a crude lactone.

Purification of the crude lactone was done by flash chromatography. Aflash column was loaded with 22 g of silica. Using 35:65(ethylacetate-hexanes), 15-20 mL fractions were collected. Combininglactone enriched fractions gave 860 mg of crude lactone. The 860 mg ofcrude lactone was dissolved in 6 mL of ethyl acetate. 2N NaOH was addedportion-wise while simultaneously monitoring HPLC for completion ofhydrolysis (<5% of lactone remaining). It required 1.3 mL of 2N NaOH forcompletion of hydrolysis (<5% of lactone remaining). pH of aq=10-11. ThepH was lowered to 3-4 by adding 0.5 mL of 2N HCl. The biphasic mixturewas stirred for 15 minutes and the layers were allowed to settle. Theorganic layer containing the product (HPLC of the aqueous layer does notshow product) was washed with 3 mL of H₂O, dried over anhydrous MgSO₄,filtered, and concentrated to yield 435 mg of(R)-2-(5-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-1-(2,3-dihydroxypropyl)-6-fluoro-1H-indol-2-yl)-2-methylpropanoicacid. LC/MS M+1=535.14.

Alternative Synthesis of(R)-2-(5-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-1-(2,3-dihydroxypropyl)-6-fluoro-1H-indol-2-yl)-2-methylpropanoicacid

Step 1. Preparation of

(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(50 g, 1.0 eq) was suspended in dichloromethane (700 mL, 14 vol) andthen cooled to −10° C. Solid carbonyl diimidazole (CDI, 34.2 g, 2.2 eq)was added. The reaction was monitored for completion by HPLC. Water (1L, 20 vol) was added to the mixture and the phases were allowed toseparate. The organic phase was solvent swapped into THF and the totalvolume was adjusted to 500 mL (10 vol). 2 M HCl (400 mL, 8 vol) wasadded to the THF solution. The mixture was stirred until all peakscoalesced into a single peak by HPLC (approximately 4 h). Toluene (700mL, 14 vol) was added to the mixture, causing phase separation. Theorganic phase was washed with water (400 mL, 8 vol). The organic phasewas concentrated at reduced pressure to give a light tan foam. The foamwas suspended in isopropyl acetate (IPA, 700 mL, 14 vol) and heated to80° C. n-Heptane (236 mL, 4.7 vol) was added at a rate to maintain thetemperature at greater than 75° C. The mixture was cooled to 20° C. at arate of 10-15° C. per hour. Crystallization occurred at approximately65° C. The mixture was then filtered. The solid was washed with 1:1IPA-heptane (120 mL, 2.4 vol) and vacuum-dried at 55° C. for 6 hours.

Step 2. Preparation of

The product from Step 1 was dissolved in dichloromethane (110 mL 20 vol)and then cooled to 10° C. N,N-Diisopropylethylamine (7.0 mL, 4 eq) wasadded to the mixture. A solution of SO₃.pyridine complex (3.3 g, 2 eq)in DMSO (11 mL, 2 vol) was then added over a period of 20 minutes, at arate to maintain internal reaction temperature between 0-10° C. When thereaction was complete based on HPLC analysis, water (55 mL, 10 vol) wasadded to the mixture at a rate to maintain the internal temp between0-10° C. Some gas evolution was observed. The reaction mixture was thenwarmed to 25° C. The phases were separated and the organic phase waswashed with 1 M HCl (220 mL, 40 vol) and then NaHCO₃ (220 mL, 40 vol).The mixture was concentrated at reduced pressure to give a white foamwhich was used without further purification.

In an alternative procedure, the product from step 1 (43.5 g, 79.1 mmol)was dissolved in toluene (305 mL, 7 vol). The solution was concentratedto remove residual IPA. The residual solid was then dissolved indichloromethane (305 mL, 7 vol). 2-Methyl-2-butene (11.2 g, 159 mmol,2.0 eq), 2,4,6-Collidine (19.3 g, 159 mmol, 2.0 eq) and PhSNH-t-Bu (2.9g, 16 mmol, 0.20 eq) were added. The mixture was cooled to −5-0° C.N-Chlorosuccinimide (11.9 g, 89 mmol, 1.12 eq) was added in 0.5-1 gportions, maintaining the internal temp at less than 2° C. Once thereaction was complete, aqueous HCl (2 M, 151 mL, 3.5 vol) was added tothe mixture. The mixture was stirred for 0.5 h while warming to ambienttemperature. Agitation was stopped and the phases were separated. Theorganic phase was washed with 5% Na₂SO₃ (200 mL, 4.6 vol), and thenwater (200 mL, 4.6 vol). The organic phase was then concentrated down toan orange oil, which was taken up in isopropanol (270 mL, 6.2 vol). Themixture was heated to 71° C. to dissolve the oil and then cooled to 40°C. at a rate of approximately 10° C./h and then to 25° C. at a rate of5° C./h. The mixture was filtered using a Buchner funnel. The wet cakewas washed with isopropanol (131 mL, 3 vol). The solid product wasvacuum-dried at 65° C. overnight.

Step 3. Preparation of

The aldehyde from Step 2 was dissolved in acetone (20 vol) and themixture was cooled to 0° C. Sodium permanganate (NaMnO₄, 40% solution inwater, 1.1 eq) was added slowly. The progress of the reaction wasmonitored by HPLC. When the reaction was complete based on HPLCanalysis, water (10 vol) was added slowly as a solid precipitated out ofsolution. The mixture was filtered. The solid was washed with acetone.The combined acetone layers were concentrated to give the product as thesodium salt as an orange foam.

In an alternative procedure, the aldehyde from step 2 (35 g, 64.3 mmol)was dissolved in acetone (210 mL, 6 vol). The mixture was concentratedto remove residual IPA. The residual foam was dissolved in acetone (350mL, 10 vol) and the resulting solution was cooled to −5-0° C. NaMnO₄ (40wt %, d=1.391 g/mL, 17.22 mL, 1.05 eq) was added in 10 equal portions,keeping the mixture temperature at less than 5° C. The reaction mixturewas stirred until complete by HPLC (approximately 30 min). Water (350mL, 10 vol) and then Celite was added to the mixture slowly, controllingthe temperature. The mixture was stirred at 0° C. for 1 h and then wasfiltered through a Celite. The brown MnO₂ wet cake was washed with 1:1acetone-water (150 mL, 4.3 vol). The acetone was removed from thecombined filtrates by distillation. NaCl (approximately 17.5 g, 0.5 wteq.) was added to the aqueous phase (approximately 5 wt % in water). Theaqueous phase was extracted with 2-methyltetrahydrofuran (350 mL, 10vol). The organic phase was azeotroped dry with 2-methyltetrahydrofuranuntil a suspension is observed. The mixture was concentrated to give acrude orange oil, which was suspended in ethanol (350 mL, 10 vol) andthen stirred for 1 h. Afterwards, the mixture was filtered through a padof Celite. The cake was washed with ethanol (70 mL, 2 vol). The solventwas swapped to acetonitrile, during which time, crystallizationoccurred. The mixture was filtered using a Buchner funnel. The product,a white solid, was washed with acetonitrile (70 mL, 2 vol) wasvacuum-dried at 55° C. overnight.

Step 4. Preparation of(R)-2-(5-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-1-(2,3-dihydroxypropyl)-6-fluoro-1H-indol-2-yl)-2-methylpropanoicacid

The product of Step 3 (5 g, 8.6 mmol) was dissolved in methanol (200 mL)and sodium carbonate (Na₂CO₃, 15 g, 17 eq) was added. The mixture wasstirred until the reaction was complete as observed by HPLC, usuallyapproximately 24 h. The reaction mixture was filtered using a Buchnerfunnel. The solvent was switched to water and the volume was adjusted to50 mL (10 vol). Acetonitrile (50 mL, 10 vol) was added. The water in themixture was slowly azeotroped out by vacuum-distillation at 35° C.Acetonitrile was continually replaced in the still pot until solidmaterial started to precipitate. The precipitate was filtered using aBuchner funnel. Filtrations were repeated until the product was pure byHPLC. The mixture was concentrated to give a light brown foam. The solidmaterial was suspended in IPA and then was heated to 60° C. for 2 hours.The suspension was cooled back to 25° C. and then stirred for 1 hour.The mixture was filtered using a Buchner funnel and the cake was washedwith IPA (10 mL, 2 vol). The solids were vacuum-dried at 60° C. for atleast 24 hours, or until the IPA content was less than 0.5 weightpercent by ¹H-NMR analysis.

In an alternative procedure, the Na salt of the product of step 3 (17.5g, 28 mmol, 1.0 eq) was dissolved in methanol (105 mL, 6 vol) andahydrous sodium carbonate (15.1 g, 142 mmol, 5 eq) was added. Thereaction mixture was filtered through a pad of Celite. The filter cakewas washed with methanol (35 mL, 2 vol). The mixture was concentrateddown to a final mass of 63 g. Acetonitrile (53 mL, 3 vol) was added tothe mixture. The hazy solution was filtered using a Buchner funnel togive a clear solution. The mixture was distilled down to half-volume.Acetonitrile (70 mL, 4 vol) was added slowly to the mixture—the productcrystallized out within approximately 5 min. The last two steps can berepeated 1-2 additional times as needed. The mixture was then stirredfor no less than 2 h. The white slurry was filtered using a Buchnerfunnel. The filter cake was washed with acetonitrile (35 mL, 2 vol). Thesolid product, the sodium salt, was vacuum-dried at 55° C. overnight.

Table 1 below recites analytical data for1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-((4R)-8-fluoro-2-hydroxy-4-(hydroxymethyl)-1,1-dimethyl-1,2,4,5-tetrahydro-[1,4]oxazepino[4,5-c]indol-9-yl)cyclopropanecarboxamide.

TABLE 1 LC/MS LC/RT M + 1 min NMR 519.20 12.54 1H NMR (501 MHz, DMSO) □7.50 (bs, 1H), 7.44-7.34 (m, 2H), 7.30 (d, J = 8.2 Hz, 1H), 7.17 (d, J =11.5 Hz, 1H), 6.51 (bs, 1H), 6.21 (s, 1H), 4.96 (m, 1H), 4.77 (d, J =2.5 Hz, 1H), 4.49 (d, J = 14.2 Hz, 1H), 4.08 (m, 1H), 3.95 (m, 1H), 3.53(m, 2H), 1.44 (t, J = 3.2 Hz, 2H), 1.34 (s, 3H), 1.28 (s, 2H), 1.10 (t,J = 3.2 Hz, 2H).

Assays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompounds

Membrane potential optical methods for assaying ΔF508-CFTR modulationproperties of compounds.

The assay utilizes fluorescent voltage sensing dyes to measure changesin membrane potential using a fluorescent plate reader (e.g., FLIPR III,Molecular Devices, Inc.) as a readout for increase in functionalΔF508-CFTR in NIH 3T3 cells. The driving force for the response is thecreation of a chloride ion gradient in conjunction with channelactivation by a single liquid addition step after the cells havepreviously been treated with compounds and subsequently loaded with avoltage sensing dye.

Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format wasdeveloped. Assay Plates containing cells are incubated for ˜2-4 hours intissue culture incubator at 37° C., 5% CO₂, 90% humidity. Cells are thenready for compound exposure after adhering to the bottom of the assayplates.

The cells were incubated in serum-free medium for 16-24 hrs in tissueculture incubator at 37° C., 5% CO₂, 90% humidity in the presence orabsence (negative control) of test compound. The cells were subsequentlyrinsed 3× with Krebs Ringers solution and loaded with a voltage sensingredistribution dye. To activate ΔF508-CFTR, 10 μM forskolin and the CFTRpotentiator, genistein (20 μM), were added along with Cl⁻-free medium toeach well. The addition of Cl⁻-free medium promoted Cl⁻ efflux inresponse to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using voltage sensor dyes.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. This HTS assay utilizes fluorescent voltagesensing dyes to measure changes in membrane potential on the FLIPR IIIas a measurement for increase in gating (conductance) of ΔF508 CFTR intemperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force forthe response is a Cl⁻ ion gradient in conjunction with channelactivation with forskolin in a single liquid addition step using afluoresecent plate reader such as FLIPR III after the cells havepreviously been treated with potentiator compounds (or DMSO vehiclecontrol) and subsequently loaded with a redistribution dye. Solutions:

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

Chloride-free bath solution: Chloride salts in Bath Solution #1 aresubstituted with gluconate salts.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, p-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at ˜20,000/well in 384-wellmatrigel-coated plates and cultured for 2 hrs at 37° C. before culturingat 27° C. for 24 hrs. for the potentiator assay. For the correctionassays, the cells are cultured at 27° C. or 37° C. with and withoutcompounds for 16-24 hours. Electrophysiological Assays for assayingΔF508-CFTR modulation properties of compounds.

1. Using Chamber Assay

Using chamber experiments were performed on polarized airway epithelialcells expressing ΔF508-CFTR to further characterize the ΔF508-CFTRmodulators identified in the optical assays. Non-CF and CF airwayepithelia were isolated from bronchial tissue, cultured as previouslydescribed (Galietta, L. J. V., Lantero, S., Gazzolo, A., Sacco, O.,Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In Vitro Cell. Dev.Biol. 34, 478-481), and plated onto Costar® Snapwell™ filters that wereprecoated with NIH3T3-conditioned media. After four days the apicalmedia was removed and the cells were grown at an air liquid interfacefor >14 days prior to use. This resulted in a monolayer of fullydifferentiated columnar cells that were ciliated, features that arecharacteristic of airway epithelia. Non-CF HBE were isolated fromnon-smokers that did not have any known lung disease. CF-HBE wereisolated from patients homozygous for ΔF508-CFTR.

HBE grown on Costar® Snapwell™ cell culture inserts were mounted in anUsing chamber (Physiologic Instruments, Inc., San Diego, Calif.), andthe transepithelial resistance and short-circuit current in the presenceof a basolateral to apical Cl⁻ gradient (I_(SC)) were measured using avoltage-clamp system (Department of Bioengineering, University of Iowa,Iowa). Briefly, HBE were examined under voltage-clamp recordingconditions (V_(hold)=0 mV) at 37° C. The basolateral solution contained(in mM) 145 NaCl, 0.83 K₂HPO₄, 3.3 KH₂PO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10Glucose, 10 HEPES (pH adjusted to 7.35 with NaOH) and the apicalsolution contained (in mM) 145 NaGluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).

Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM), PDE inhibitor, IBMX (100 μM) and CFTR potentiator,genistein (50 μM) were added to the apical side.

As observed in other cell types, incubation at low temperatures of FRTcells and human bronchial epithelial cells isolated from diseased CFpatients (CF-HBE) expressing ΔF508-CFTR increases the functional densityof CFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with test compound for 24-48 hoursat 37° C. and were subsequently washed 3× prior to recording. The cAMP-and genistein-mediated I_(SC) in compound-treated cells was normalizedto 37° C. controls and expressed as percentage activity of CFTR activityin wt-HBE. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(SC) comparedto the 37° C. controls.

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge CF concentration gradient across the epithelium. Forskolin (10 μM)and all test compounds were added to the apical side of the cell cultureinserts. The efficacy of the putative ΔF508-CFTR potentiators wascompared to that of the known potentiator, genistein.

2. Patch-clamp Recordings

Total Cl⁻ current in ΔF508-NIH3T3 cells was monitored using theperforated-patch recording configuration as previously described (Rae,J., Cooper, K., Gates, P., & Watsky, M. (1991) J. Neurosci. Methods 37,15-26). Voltage-clamp recordings were performed at 22° C. using anAxopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City,Calif.). The pipette solution contained (in mM) 150 N-methyl-D-glucamine(NMDG)-Cl, 2 MgCl₂, 2 CaCl₂, 10 EGTA, 10 HEPES, and 240 μg/mlamphotericin-B (pH adjusted to 7.35 with HCl). The extracellular mediumcontained (in mM) 150 NMDG-Cl, 2 MgCl₂, 2 CaCl₂, 10 HEPES (pH adjustedto 7.35 with HCl). Pulse generation, data acquisition, and analysis wereperformed using a PC equipped with a Digidata 1320 A/D interface inconjunction with Clampex 8 (Axon Instruments Inc.). To activateΔF508-CFTR, 10 μM forskolin and 20 μM genistein were added to the bathand the current-voltage relation was monitored every 30 sec.

Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 μM forskolin and 20 μM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 μM of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 μM of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in IΔ_(F508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1×pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

3. Single-Channel Recordings

Gating activity of wt-CFTR and temperature-corrected ΔF508-CFTRexpressed in NIH3T3 cells was observed using excised inside-out membranepatch recordings as previously described (Dalemans, W., Barbry, P.,Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G.,Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-528)using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.).The pipette contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl₂, 2MgCl₂, and 10 HEPES (pH adjusted to 7.35 with Tris base). The bathcontained (in mM): 150 NMDG-Cl, 2 MgCl₂, 5 EGTA, 10 TES, and 14 Trisbase (pH adjusted to 7.35 with HCl). After excision, both wt- andΔF508-CFTR were activated by adding 1 mM Mg-ATP, 75 nM of the catalyticsubunit of cAMP-dependent protein kinase (PKA; Promega Corp. Madison,Wis.), and 10 mM NaF to inhibit protein phosphatases, which preventedcurrent rundown. The pipette potential was maintained at 80 mV. Channelactivity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

In Table 2, the following meanings apply:

EC50: “+++” means <2 uM; “++” means between 2 uM to 5 uM; “+” meansbetween 5 uM to 25 uM.

% Efficacy: “+” means <25%; “++” means between 25% and 100%; “+++” means>100%.

TABLE 2 Compound EC50 % Efficacy 1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-((4R)- +++ +++ 8-fluoro-2-hydroxy-4-(hydroxymethyl)-1,1-dimethyl-1,2,4,5-tetrahydro-[1,4]oxazepino[4,5-α]indol-9-yl)cyclopropane carboxamide

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein independently foreach occurrence: Y is OH or NH; and X is CO₂J; wherein J is H or C₁-C₆alkyl; R is H, OH, OCH₃ or two R taken together form —OCH₂O— or —OCF₂O—;R₁ is H or up to two C₁-C₆ alkyl; R₂ is H or halo; and R₃ is H or C₁-C₆alkyl; or Y and X combine to form a compound of formula II:

or a pharmaceutically acceptable salt thereof, wherein independently foreach occurrence: R is H, OH, OCH₃ or two R taken together form —OCH₂O—or —OCF₂O—; R₁ is H or up to two C₁-C₆ alkyl; R₂ is H or halo; R₃ is Hor C₁-C₆ alkyl; Y is O or NR₄, and R₄ is H or C₁-C₆ alkyl.
 2. Thecompound of claim 1 of formula I, wherein two R taken together form—OCF₂O—, R₁ is H, and R₂ is F.
 3. The compound of claim 1 of formula I,wherein two R taken together form —OCF₂O—, R₁ is H, R₂ is F, and R₃ isCH₃.
 4. The compound of claim 1 of formula I, wherein two R takentogether form —OCF₂O—, R₁ is H, R₂ is F, R₃ is CH₃, and X is CO₂H. 5.The compound of claim 1 of formula I, wherein two R taken together form—OCF₂O—, R₁ is H, R₂ is F, R₃ is CH₃, X is CO₂H, and Y is OH.
 6. Thecompound of claim 1 of formula II, wherein two R taken together form—OCF₂O—, R₁ is H, and R₂ is F.
 7. The compound of claim 1 of formula II,wherein two R taken together form —OCF₂O—, R₁ is H, R₂ is F, and R₃ isCH₃.
 8. The compound of claim 1, having formula IIa:

or a pharmaceutically acceptable salt thereof, wherein: R₂ is H or halo.9. The compound of claim 8, wherein R₂ is F.
 10. The compound of claim1, wherein the compound is


11. The compound of claim 1, wherein the compound is


12. The compound of claim 1, wherein the compound is


13. A pharmaceutical composition comprising (i) a compound according toclaim 1; and (ii) a pharmaceutically acceptable carrier.
 14. Thecomposition of claim 13, further comprising an additional agent selectedfrom a mucolytic agent, bronchodialator, an anti-biotic, ananti-infective agent, an anti-inflammatory agent, CFTR corrector, CFTRpotentiator, or a nutritional agent.
 15. A method of increasing thenumber of functional ABC transporters in a membrane of a cell,comprising the step of contacting the cell with a compound of claim 1.16. The method of claim 15, wherein the ABC transporter is CFTR.
 17. Amethod of treating a condition, disease, or disorder in a subjectimplicated by ABC transporter activity, comprising the step ofadministering to the subject a compound or composition of claims
 1. 18.The method of claim 17, wherein the condition, disease, or disorder isselected from cystic fibrosis, emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, protein C deficiency, Type 1hereditary angioedema, lipid processing deficiencies, familialhypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,lysosomal storage diseases, 1-cell disease/pseudo-Hurler,mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, diabetes mellitus, laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, diabetesinsipidus (di), neurophyseal di, neprogenic DI, Charcot-Marie Toothsyndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, polyglutamineneurological disorders, Huntington, spinocerebullar ataxia type I,spinal and bulbar muscular atrophy, dentatorubal pallidoluysian,myotonic dystrophy, spongiform encephalopathies, hereditaryCreutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome,COPD, dry-eye disease, or Sjögren's disease.
 19. The method of claim 18,wherein the condition, disease, or disorder is selected from cysticfibrosis, emphysema, COPD, or dry-eye disease.
 20. A kit for use inmeasuring the activity of a ABC transporter or a fragment thereof in abiological sample in vitro or in vivo, comprising: (i) a compoundaccording to claims 1; and (ii) instructions for: a) contacting thecompound with the biological sample; and b) measuring activity of saidABC transporter or a fragment thereof.
 21. The kit according to claim20, further comprising instructions for a) contacting an additionalcompound with the biological sample; b) measuring the activity of saidABC transporter or a fragment thereof in the presence of said additionalcompound, and c) comparing the activity of the ABC transporter in thepresence of the additional compound with the density of the ABCtransporter in the presence of the first compound.
 22. A process forpreparing a compound of formula Ia

comprising converting an ester of formula I-1 to a compound of formulaIa:

wherein independently for each occurrence: R₂ is H or halo; and R₄ isC₁-C₆ alkyl or benzyl.
 23. The process of claim 22, wherein R₂ is H orF, and R₄ is methyl, ethyl, isopropyl, butyl, or benzyl.
 24. The processof claim 23, wherein R₂ is H or F, and R₄ is isopropyl or benzyl. 25.The process of claim 22, wherein converting comprises contacting thecompound of formula I-1 with a base in the presence of a solvent. 26.The process of claim 25, wherein the base is an alkali or alkali metalhydroxide. In one embodiment, the base is NaOH or LiOH and the solventis methanol or THF either of which may be admixed with water.
 27. Aprocess for preparing a compound of formula Ia

wherein R₂ is H or halo, comprising: (a) contacting the compound offormula I-45 with carbonyl diimidazole (CDI) in the presence of asolvent as provided above to give a compound of formula I-4

(b) contacting the compound of formula I-4 with an oxidant in thepresence of a solvent as provided above to give a compound of formulaI-3

(c) contacting the compound of formula I-3 with an oxidant in thepresence of a solvent as provided above to give compound of formula I-2;

and (d) contacting the compound of formula I-1 with a base in thepresence of a solvent as provided above to give a compound of formulaIa.


28. The process of claim 27, wherein R₂ is H or F.
 29. A process forpreparing a compound of formula IIa

wherein R₂ is H or halo, comprising: (a) contacting the compound offormula I-5 with carbonyl diimidazole (CDI) in the presence of a solventas provided above to give a compound of formula I-4;

(b) contacting the compound of formula I-4 with an oxidant in thepresence of a solvent as provided above to give a compound of formulaI-3;

and (c) contacting the compound of formula I-3 with a base in thepresence of a solvent as provided above to give a compound of formulaIIa;


30. The process of claim 29, wherein R₂ is H or F.
 31. A compound whichis:

wherein R₂ is H or F and R₄ is iPr or benzyl.
 32. The compound of claim30 which is:

wherein R₂ is H or halo and R₄ is iPr or benzyl.
 33. The compound ofclaim 30 which is:


34. The compound of claim 30 which is: